Methods and system for interfering with viability of bacteria and related compounds and compositions

ABSTRACT

Provided herein are methods and systems for interfering with viability of bacteria and related compounds and compositions.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 13/456,172, filed on Apr. 25, 2012, which in turn, claimspriority to U.S. Provisional Application No. 61/478,746 filed on Apr.25, 2011 and entitled “Methods and systems for interfering withviability of Bacteria and Related Compounds and compositions”, each ofwhich is incorporated herein by reference in its entirety.

FIELD

The present disclosure relates to methods and systems for interferingwith viability of bacteria and related compounds and compositions.

BACKGROUND

Bacteria viability has been the focus of research in the field ofbiological analysis, in particular when aimed at medical applicationssuch as therapeutic or diagnostic application.

Whether for pathological examination or for fundamental biology studies,several methods are commonly used for the detection and interferencewith the viability of bacteria.

Although various methods, systems and compositions have been developedto interfere, and in particular, reduce bacterial viability to theextent of killing the bacteria, antibiotic resistance and additionaldefense mechanisms of the microorganism have made development ofmethods, systems and compositions able to interfere and in particularinhibit bacterial viability particularly challenging.

SUMMARY

Provided herein, are methods and systems and related compounds andcompositions that in several embodiments are suitable for reducingantibiotic resistance and/or survivability of bacteria. In severalembodiments, compositions, methods and systems herein described areexpected to be suitable to treat and/or prevent bacterial infection invitro or in vivo.

According to a first aspect, a method and system to interfere withviability of bacteria is described, the method comprising inactivating aphenazine and/or a phenazine related pathway in the bacteria to reducesurvivability and/or antibiotic resistance of the bacteria. The systemcomprises one or more agents able to inactivate a phenazine and/or aphenazine related pathway in the bacteria and an antibiotic and/or otherantimicrobial. In some embodiments of the methods and systems, thebacteria comprise persister cells.

According to a second aspect, a method and system to inactivate abacterium is described. The method comprises contacting the bacteriumwith an agent capable of inactivating a phenazine and/or a phenazinerelated pathway in the bacterium in combination with an antibioticand/or other antimicrobial. The system comprises one or more agents ableto inactivate a phenazine and/or a phenazine related pathway in thebacteria and an antibiotic and/or other antimicrobial. In someembodiments of the methods and systems, the bacteria comprise persistercells.

According to a third aspect, a method and system for treating and/orpreventing a bacterial infection in an individual is described. Themethod comprises administering an effective amount of one or more agentsable to specifically inactivate a phenazine and/or a phenazine relatedpathway in the bacteria. In particular, in some embodiments,administering of one or more agents can be performed in combination withone or more antibiotics and/or other antimicrobials. The systemcomprises one or more agents able to specifically inactivate a phenazineand/or a phenazine related pathway in the bacteria and an antibioticand/or other antimicrobial. In some embodiments of methods and systems,the bacteria comprise persister cells.

According to a fourth aspect, a method and system for identifying anantimicrobial is described. The method comprises contacting a microbewith a candidate agent and detecting the ability of the candidate agentof inactivating a phenazine and/or a phenazine related pathway in thebacteria. The system comprises one or more microbes and one or moreagents capable of detecting phenazine and/or phenazine related pathways.In some embodiments of the methods and systems, the bacteria comprisepersister cells.

According to a fifth aspect, an antimicrobial is described. Theantimicrobial comprises one or more agents able to inactivate aphenazine and/or a phenazine related pathway in the bacteria. The one ormore agents are in particular comprised in the antimicrobial in anamount suitable to reduce antibiotic resistance and/or survivability ofbacteria. In some embodiments, the antimicrobial comprises a compatiblevehicle, which can be a vehicle for effective administrating and/ordelivering of the one or more agents to an individual. In someembodiments of the methods and systems, the bacteria comprise persistercells.

According to a sixth aspect, a method and system to interfere with theviability of bacteria in a medium is described, the method comprisessubtracting from the medium Fe(II) alone or in combination with Fe(III)to reduce survivability and/or antibiotic resistance of the bacteria.The system comprises one or more agents capable of subtracting Fe(II)and/or Fe(III) for simultaneous combined or sequential use in the methodherein described. In some embodiments, the subtracting is performed bysubtracting Fe(II). In other embodiments, the subtracting is performedby subtracting Fe(II) and Fe(III) from the medium.

According to a seventh aspect an antimicrobial is described. Theantimicrobial comprises one or more agents able to subtract Fe(II) froma medium suitable to host bacteria. The one or more agents are inparticular comprised in the antimicrobial in an amount suitable toreduce antibiotic resistance and/or survivability of bacteria. In someembodiments, the antimicrobial comprises a compatible vehicle, which canbe a vehicle for effective administrating and/or delivering of the oneor more agents to an individual. In some embodiments of the methods andsystems, the bacteria comprise persister cells.

The methods and systems herein described, and related compounds andcompositions in several embodiments allow reducing antibiotic resistanceand/or bacterial survivability according to distinct mechanism andpathways wherein phenazine functions.

The methods and systems and related compounds and compositions hereindescribed can be used in connection with applications wherein reductionof viability of bacteria and/or reduction of antibiotic resistance isdesired, which include but are not limited to medical application, drugresearch, biological analysis and diagnostics including but not limitedto clinical applications.

The details of one or more embodiments of the disclosure are set forthin the accompanying drawings and the description below. Other features,objects, and advantages will be apparent from the description anddrawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated into and constitute apart of this specification, illustrate one or more embodiments of thepresent disclosure and, together with the detailed description andexamples sections, serve to explain the principles and implementationsof the disclosure.

FIG. 1. The phenazine PCA can work together with the siderophorepyoverdin in promoting P. aeruginosa biofilm formation. (A) Confocalmicroscopic images of biofilms at day 4, (B) total biomass over time asinferred by COMSTAT analysis, and (C) the corresponding release ofphenazine(s) and/or siderophore(s) into the biofilm effluents forYFP-labeled PA14 wild type (WT), the phenazine null strain (Δphz), thesiderophore null strain (ΔpvdAΔpchE), and the phenazine-siderophore nullstrain (ΔphzΔpvdAΔpchE) under a flow of 1% TSB medium at 22° C. Confocalimages consist of top-down views (x-y plane, top images) and side views(x-z plane, bottom images; enlarged and truncated to emphasizedifferences in the z dimension). Scale bars: 100 μm for top-down viewimages, 50 μm for side-view images. Results are representative of 6experiments. Data reported in (B) and (C) represent the mean±SD. Relatedquantitative data can be found in Table 3.

FIG. 2. PCA and PYO can circumvent the siderophore pathway for promotingP. aeruginosa biofilm development via Fe(II) uptake dependent andindependent mechanisms, respectively. The effectively insoluble Fe(III)mineral ferrihyrite [Fe(OH)₃(s)] was the Fe(III) source. Confocalmicroscopic images of YFP-labeled P. aeruginosa PA14 siderophore nullstrain (ΔpvdAΔpchE) incubated in biofilm flow cells at 22° C. for 6 dayswith no addition, or with addition of 1.0 μM Fe(OH)₃(s), 10 μM phenazine(PCA or PYO), or 1.0 μM Fe(OH)₃(s) together with 10 μM phenazine (PCA orPYO), to 1% TSB medium. Images are top-down views (x-y plane); scalebar: 100 μm. Results are representative of 4 experiments. Relatedquantitative data can be found in Table 4.

FIG. 3. The P. aeruginosa PA14 feoB::MAR2xT7 mutant (feoB::tn) cannotgrow when given Fe(II) as its sole iron source, yet can grow in thepresence of Fe(III); the wild type (WT) can grow regardless of whetheriron was in the ferric or ferrous form. Cells were incubated shakinganaerobically in Amberlite-treated 1% TSB medium containing 100 mM KNO₃,50 mM glutamate, 1% glycerol, and 100 μM iron source (either(NH₄)₂Fe(II)(SO₄)₂ or Fe(III)Cl₃) at 37° C. for 22 hours. Data reportedare the mean of triplicate experiments±SD.

FIG. 4. A. The presence of sub-inhibitory levels of conalbumin alone ortogether with phenazine (PCA or PYO) does not inhibit planktonic growthof P. aeruginosa PA14 strains. Experiments were performed in batchcultures in 1% TSB-based biofilm medium at 37° C. Data reported are themean of triplicate experiments±SD. B. PCA but not PYO can rescue theconalbumin-induced P. aeruginosa biofilm defect by reducingprotein-sequestered Fe(III) with concomitant release of Fe(II). Confocalmicroscopic images of YFP-labeled P. aeruginosa PA14 wild type (WT), andDIC microscopic images of the P. aeruginosa PA14 feoB::MAR2xT7 mutant(feoB::tn) disrupted in Fe²⁺ transport into the cytoplasm, incubated inbiofilm flow cells at 22° C. for 6 days with no addition, with additionof 40 μg/ml iron-free conalbumin alone, or together with 10 μM phenazine(PCA or PYO), to 1% TSB media flow. Confocal images consist of top-downviews (x-y plane, top images) and side views (x-z plane, bottom images;enlarged and truncated to emphasize differences in the z dimension). DICimages are top-down views (x-y plane). Scale bars: 100 μm for top-downview images, 50 μm for side-view images. Results are representative of 4experiments. Related quantitative data can be found in Table 5.

FIG. 5. Summary showing phenazine-facilitated Fe(II) uptake (on theright) in contrast to Fe(III) uptake (on the left). Fe(III) uptake isdrawn in a highly simplified manner where the star depicts asiderophore, e.g. pyoverdin. The siderophore binds extracellular Fe(III)and crosses the outer membrane (OM) via a TonB-dependent transporter. Inthe periplasm, Fe(III) is released from the siderophore, which can thenbe recycled; Fe(III) is reduced by an unknown mechanism to Fe(II) in theperiplasm (P) and transported across the cytoplasmic membrane (CM)presumably by an ABC transport system. In contrast, phenazines canreduce extracellular Fe(III) to Fe(II). After entering the periplasm,presumably via an OM porin, Fe(II) is transported across the CM viaFeoB. Phenazines themselves are recycled, and enter and leave the cellthrough various transporters (not drawn for simplicity).Intracellularly, phenazine reduction is coupled to NADH oxidation toNAD⁺, although whether this reduction is enzyme-mediated is unknown.Reduced phenazine is indicated by the open oval; oxidized phenazine bythe filled oval.

FIG. 6. Δphz1/2 has increased swarming motility. Quantification ofswarming motility. Images of three swarming plates for each strain orcondition were captured, exported to Adobe Photoshop and the agarsurface covered by the swarms was quantified using the Analysis Tools.

FIG. 7. Phenazines support development of structured biofilms in acontinuous flow cell-system. Representative images of 4-day old biofilmsof the wild-type (A) and phz1/2 mutant without (B) and with addition of25 μM pyocyanin (C) to the nutrient flow. Images of similar positions inthe flow cell were taken in triplicate. Three independent experimentswere performed with similar results.

FIG. 8. Phenazines modulate colony-biofilm morphology. (A, B) P.aeruginosa PA14 wild-type and Δphz1/2 cultures were spotted onto 1% agarplates (containing 1% tryptone, 40 μg/ml Congo Red and 20 μg/mlCoomassie Blue) and incubated at 20′C for 7 days in the presence of 0.1mM PCA, 0.1 mM or in the absence of supplements. Each day colonies fromthree independent experiments were scanned and their surface coveragewas determined. Representative images of colonies at day 2, 4 and 6 areshown (A; scale bar is 1 cm). The data in (B) show the average surfacecoverage and standard deviation for the three independent experiments.(C) Phenazine titrations. Colonies were grown as above, supplementedwith 0, 0.1, 0.2, 0.3 or 0.4 mM PCA or pyocyanin. Surface coverage ofcolonies from three independent experiments was determined afterincubation at 20′C for 7 days. Bars indicate the standard deviation.

FIG. 9. Stationary-phase P. aeruginosa PA14 cultures produce pyocyaninand directly catalyze its reduction. (A) Tube 1, exponential-phase LBculture; tube 2, stationary-phase LB culture, immediately after removalfrom a shaking incubator; tube 3, stationary-phase LB culture, leftstanding at room temperature for ˜5 minutes; tube 4, 100 μM pyocyanin inMOPS buffer, left standing at room temperature for ˜5 minutes; tube 5same culture as in tubes 2 & 3, resuspended in buffer shown in tube 4and left standing at room temperature for ˜5 minutes; tube 6, samesuspension as in tube 5, after vortexing. (B) Absorbance spectra ofbuffer and supernatants from (A), tubes 4-6. The suspension from tube 5was centrifuged and placed in a stoppered cuvette under anaerobicconditions. The pyocyanin/buffer spectrum overlaps almost completelywith that of the supernatant from the aerated culture. PYO, pyocyanin.

FIG. 10. The rate of pyocyanin reduction increases in stationary phasein P. aeruginosa PA14. A 100-ml P. aeruginosa LB culture was grown in a500-ml Erlenmeyer flask and sampled at various points in the growthcurve. Cells were concentrated or diluted in culture supernatant tonormalize their OD (600 nm) to 0.8, amended with pyocyanin, thentransferred to anaerobic cuvettes and stoppered. Absorbance at 690 nmwas measured over time and was converted to the concentration ofoxidized pyocyanin remaining in the cuvette. Gray squares indicate timepoints at which samples were taken for cell suspension assays. The blacksquare indicates the first appearance of pyocyanin in the culture. Datashown is representative of three separate experiments. OD, opticaldensity.

FIG. 11. Pyocyanin exposure effects redox balancing in stationary phasein a manner analogous to that of a known physiological electronacceptor. (A) Growth and pyocyanin production for wild type P.aeruginosa PA14 grown aerobically in 10 ml LB in 18×150 mM tubes. (B)NADH/NAD⁺ ratios for cultures grown under the same conditions as thosedescribed in part (A). At 7 hours, pyocyanin production in the wild typecultures was visible by eye. 45 μM (half the expected finalconcentration) was added to the Δphz cultures to be tested forcomplementation, and 15 mM KNO₃ was added to cultures to be tested forthe effect of an additional electron acceptor. At 9 hours, pyocyanin inthe wild type cultures had increased to near-maximum concentrations, soa second dose of pyocyanin or KNO₃ was added to the appropriatecultures, for final concentrations of 90 μM and 30 mM, respectively.Water was added to negative controls. Eleven hours after inoculation,and 2 hours after the addition of the final dose of pyocyanin, NAD(H)was extracted and assayed for each culture. (C), NADH/NAD⁺ ratios forcultures treated as in part (B), but with varying concentrations ofpyocyanin added. (D) NADH and NA⁺ concentrations for cultures describedin (B), normalized to OD (500 nm). Error bars represent the standarddeviations of triplicate samples. OD, optical density. wt, wild type.

FIG. 12. NADH accumulates in stationary phase in cultures limited foroxygen and defective in pyocyanin production. P. aeruginosa wild typeand Δphz cultures were grown in 1 L MOPS synthetic medium supplementedwith either 50 or 10 mM glucose in a 3-L fermentor with constantaeration and agitation. Cultures were sampled at various points in thegrowth curve to allow measurement of the optical density (OD) at 500 nmand extraction of NAD(H). Relative dissolved oxygen concentrations weremeasured throughout growth using a polarographic oxygen electrode. OD(500 nm), dO₂, and NADH/NAD⁺ are shown for (A), the Δphz mutant grown inmedium containing 50 mM glucose, and (B) the Δphz mutant grown in mediumcontaining 10 mM glucose. For (C), wild type P. aeruginosa PA14 grown inmedium containing 50 mM glucose, these parameters plus the concentrationof pyocyanin produced by the culture are shown.

FIG. 13. Wild type P. aeruginosa PA14 excretes pyruvate in stationaryphase, and addition of pyocyanin to Δphz mutant cultures restores thepyruvate excretion phenotype. Cultures were inoculated into MOPSsynthetic medium amended with 50 mM glucose (initial OD (500 nm)=0.03).To complement pyruvate excretion, 50 μM pyocyanin was added to the Δphzculture at the time when pyocyanin reached its maximum concentration inthe wild type cultures (approximately 12 hours after inoculation). 20 μlof culture filtrates at the 24-hour time point were loaded onto an anionexchange column and subjected to an isocratic gradient in 5 mM H₂SO₄.Pyruvate peaks are indicated by arrows. The elution time of pyruvatedrifts slightly but averages around 10.5 minutes. Results shown arerepresentative of three separate experiments. Other peak identities aredescribed in the text.

FIG. 14. P. aeruginosa PA14 cultures consume excreted pyruvate in verylate stationary phase. Duplicate cultures were inoculated at OD (500nm)˜0.001 in MOPS synthetic medium amended with 50 mM glucose.Approximately every 4 hours, 100-200 μl culture were sampled andfiltered for HPLC analysis as described for FIG. 4. (A), OD 500 for wildtype and Δphz cultures plotted on a logarithmic scale. (B), same data asin (A) plotted on a linear scale to show the lower growth yieldsconsistently observed for wild type PA14 under this condition. (C)Quantification of pyruvate production for the “wt 1” and “wt 2”cultures, and inset, chromatograms demonstrating the disappearance ofpyruvate at 42 hours for the “wt 1” culture. The arrow indicates theelution time of the pyruvate peak. wt, wild type.

FIG. 15. Model: pyocyanin reduction allows P. aeruginosa PA14 tomaintain redox homeostasis under oxygen-limited conditions. Whensufficient oxygen is available for growth (A), the aerobic respiratorychain (“resp”) can catalyze the reoxidation of NADH. Under conditions inwhich terminal electron acceptors for respiration are limiting (B), P.aeruginosa can couple the re-oxidation of NADH to the reduction ofpyocyanin, either directly or through an enzyme-mediated reaction asrepresented by “pyocyanin red,” a putative phenazine reductase. Theelectrons could be transferred from pyocyanin to oxygen through anabiotic extracellular reaction. (C) Also under conditions of oxygenlimitation, the NADH/NAD⁺ ratio could be balanced through inactivationof the pyruvate dehydrogenase complex by pyocyanin. NAD⁺ reduction (andtherefore NADH production) would be avoided because pyruvate would beexcreted without further oxidation.

FIG. 16. A) Cross-sectional analysis of sputum pyocyanin concentrationsreveals a positive correlation with pulmonary function decline (FEV1%).Hybridization chain reaction imaging of (B) wild type P. aeruginosa and(C) a mutant containing a deletion of bqsR, reveals differential geneexpression patterns of the genes bqsS (green) and bqsR (red) in responseto 50 μM Fe(II) (left) and no treatment (right).

FIG. 17 Sputum iron chemistry versus disease severity. Reported valuesare mean concentrations+/−one standard deviation, and are conservativeestimates based on3-(2-Pyridyl)-5,6-diphenyl-1,2,4-triazine-p,p′-disulfonic acidmonosodium salt hydrate, which is also known under the trademarkFerrozine® and ICP-MS measurements. (B) Biofilm growth prevention(white) and dissolution (black) by conalbumin (an Fe(III)-chelator) andFerrozine® (an Fe(II)-chelator). Effects are mitigated by addition of 80μM Fe(II). Symbols represent significance versus (*) untreated controlsand (●) combination chelator treatment. Error bars represent thestandard error of the mean (n=18).

FIG. 18 Determination of total sputum iron using ICP-MS versusFerrozine®. The latter approach generally estimates ˜30% more total ironthan the more sensitive mass spectrometry method. Dashed line representsthe 1:1 trendline.

FIG. 19: Schematic illustration of a Model of the QS network in P.aeruginosa PA14. The PA14 QS from Dietrich et al. (MolecularMicrobiology 2006, 61, 1308-1321)

DETAILED DESCRIPTION

Provided herein are methods and systems that are based on inactivationof phenazine and/or a phenazine related pathway.

The term “phenazine” as used herein indicates small, colorful,redox-active compounds formed by bacteria to perform diversephysiological functions. Well over 50 phenazines of bacterial origin areknown. Among these phenazines, only a few of which have been giventrivial names, represent every color of the visible spectrum. Theabsorption spectra of phenazines are characteristic, with an intensepeak in the range 250-290 nm and a weaker peak at 350-400 nm. At leastone main band occurs in the visible region (400-600 nm) to which thephenazines owe their colors.

The phenazine pigments are mostly water soluble and are excreted intothe medium. For example, pyocyanin produced by Pseudomonas aeruginosa,diffuses readily into agar-solidified media which become stained blue.Some phenazines are only sparingly water soluble and precipitate. Forexamples, chlororaphine, a mixture of phenazine-1-carboxamide(oxychlororaphine) and its dihydro derivative, produced by Pseudomonaschlororaphis, accumulate as isolated emerald-green crystals at the baseof agar slants. Iodinin crystallizes on the surfaces of old colonies ofBrevibacterium iodinum, giving them a dark-purple appearance, andphenazine-1-carboxylic acid (PCA) is deposited as golden yellow crystalsin colonies of Pseudomonas aureofaciens and in the surrounding medium.

Considerable progress has been made in elucidating the biosynthesis aswell as properties of individual phenazines. Examples are provided belowfor two types of phenazines known as pyocyanin andphenazine-1-carboxylic acid, respectively. For more examples of theoccurrence, biochemistry and physiology of phenazine production, seeTurner et al., 1986, Advances in Microbial Physiology, vol. 27, page211-275.

Pyocyanin is the phenazine characteristically produced by chromogenicstrains of the pseudomonad, which is found as the blue pigmentoccasionally seen on infected wound dressings. More attention has beenpaid to pyocyanin than to any other phenazine. Pyocyanin is an organicbase, blue in alkaline aqueous solutions but red when acidified. Thedifferential solubility of these forms in chloroform and water wasexploited for this pigment. Pyocyanin was found to be chemically reducedto a colorless form and spontaneously reoxidized in air, which has ledto the discovery, the indicator and redox properties of the compound.Additionally, pyocyanin slowly decomposed to a yellow substance, nolonger basic in nature, now known to be 1-hydroxyphenazine.

Phenazine-1-carboxylic acid (PCA) is a yellow crystalline compoundnaturally produced by P. aureofaciens. The phenazine produced wasreadily extracted from acidified cultures with chloroform. Dilute alkalichanged the color of the phenazine to orange-red and rendered itinsoluble in chloroform. PCA isolated from cultures, in amounts of up to1 g of pigment litre⁻¹, was shown to have antibacterial activity towardsa number of plant pathogens.

Phenazines can also include, but are not limited to, molecules accordingto the structure and formula below:

where R₁-R₈ are independently selected from hydrogen, hydroxy, alkoxy,alkyl, alkenyl, alkynyl, aryl, heteroaryl, acyl, and other groupsidentifiable to the skilled person.

Additionally, phenazines can include, but are not limited to, moleculesaccording to the structures and formulas below:

where R₁-R₁₀ are independently selected from hydrogen, hydroxy, alkoxy,alkyl, alkenyl, alkynyl, aryl, heteroaryl, acyl, and other groupsidentifiable to the skilled person, and one of R₁-R₁₀ is a negativelycharged substituent (formal charge of −1) such as

In particular, exemplary phenazine structures comprise:

as well as additional phenazines that can be identified by a skilledperson such as the exemplary phenazines described in Mentel et al.(ChemBioChem 2009, 10, 2295-2304) and Pierson et al. (Appl Microbiol.Biotechnol. 2010, 86, 1659-1670)

Production of phenazines is of obvious taxonomic value, particularlywhen relatively few genera are concerned. It should be noted, however,that the same pigment may be produced by unrelated bacteria and“achromogenic” strains of many phenazine-producers are common. Also, anumber of strains of bacteria produce more than one phenazine. And itseems likely that all bacterial phenazines are derived from a commonprecursor.

Recent study has uncovered diverse physiological functions of phenazinesfor the cells that produce them. For example, the opportunistic pathogenPseudomonas aeruginosa is the dominant pathogen that infects individualswith cystic fibrosis (CF). Production of pyocyanin by P. aeruginosa isresponsible for the bluish tint of sputum and pus associated with P.aeruginosa infections in humans. Clear correlation has been demonstratedbetween phenazine concentration in sputum and lung function decline.Further, phenazines are found to affect bacterial community developmentfor P. aeruginosa.

Several aspects of the disclosure relate, at least in part, to thediscovery that phenazines function to promote antibiotic resistance andsurvivability of bacteria through various distinct mechanisms andpathways and that the related inactivation affect viability of thebacteria.

The term “inactivation” as used herein with reference to a pathwayrefers to a complete or partial inhibition of one or more of thereactions or steps in the pathway.

The terms “inhibit” and “inhibition” as used herein refers to a decreaserelative to a baseline level. Accordingly, inhibition of a reactionindicates a decrease in the relative output compared to an outputselected as a baseline level. Inhibition of a reaction can be detectedby detecting any products or other indicator and/or parameter associatedwith completion of the reaction and identifiable by a skilled person.Exemplary inhibition of a reaction can be performed by contactingenzymes catalyzing the reaction with a suitable enzyme inhibitor (i.e. amolecule that decreases the enzyme activity) such that lesser quantitiesof intermediates and/or products are produced relative to a baselinelevel. Exemplary inhibition of a step or reaction can also be performedby removal of the relevant substrate and/or intermediate substance (suchas, for example, subtraction of Fe(III) and/or Fe(II) in the Fe(III)reduction to Fe(II) and Fe(II) importation pathways), by removing anenzyme involved in the reaction (such as, for example, by suppressingthe expression of one of the genes coding for the enzyme), by removing areducing agent and/or starting material from an oxidation-reductionreaction such that less product is produced, by removing an iontransporter and/or pre-transported ion such that less of the ion istransported into a bacteria, and other approaches and techniquesidentifiable to a skilled person upon reading of the present disclosure.Accordingly an inactivated pathway in the sense of the presentdisclosure indicates a pathway in which any enzyme controlling areaction in the pathway is biologically inactive or in which at leastone of the reactions or steps of the pathway is otherwise inhibited,e.g. by deleting one or more genes encoding for enzymes of the pathwayand/or by subtracting the relevant substrate and/or intermediate.

The term “pathway” as used herein refers to a biological processcomprising one or more chemical or biological reactions or steps inwhich at least one substance is transformed, produced, and/or acquiredby a bacteria. The one or more reactions or steps comprised in thepathway can involve molecules such as, for example, proteins, enzymes,cofactors, oxidizing/reducing agents, signaling molecules, metal ions,and others identifiable to a skilled person upon reading of the presentdisclosure that participate in the transformation, production and/oracquisition of the substance by a bacteria. In embodiments whereinpathway involves a bacterial cell signaling molecule, the pathwayindicates signal transduction through cascade reactions of a series ofsignaling molecules as part of a complex system of communication thatgoverns basic cellular activities and coordinates cell actions.Exemplary pathways of the disclosure include, but are not limited to,phenazine biosynthesis comprising the steps of synthesizing phenazinesfrom various starting materials (such as, for example,erythrose-4-phosphate, phosphoenol pyruvate, and/or other startingmaterials) through the use of enzymes (such as, for example, PhzA, PhzB,PhzE, PhzD, and others), Fe(III) reduction to Fe(II) comprising thesteps of reduction of Fe(III) to Fe(II) through the use of a reducingagent (such as, for example, phenazine-1-carboxylate, pyocyanin, and/orother reducing agents), bacterial acquisition of Fe(II) comprising thesteps of reduction of Fe(III) to Fe(II) by a reducing agent (such as,for example, phenazine-1-carboxylate, pyocyanin, and/or other reducingagents) and importation of Fe(II) into the bacteria by a transporterprotein (such as, for example, FeoB), and other pathways identifiable toa skilled person upon reading of the present disclosure.

The term “phenazine-related pathway” as used herein refers to either apathway in which a phenazine is a starting material, intermediate, orproduct, or alternatively, any pathway in which at least one of the oneor more of the steps comprised in the pathway are mediated by aphenazine. Exemplary pathways in which a phenazine is a startingmaterial, intermediate, or product include, but are not limited to,phenazine biosynthesis, phenazine cycling, quorum sensing, and otherpathways identifiable to a skilled person upon reading of the presentdisclosure. Exemplary pathways in which one or more of the steps of thepathway are promoted or mediated by a phenazine include, but are notlimited to, reduction of Fe(III) to Fe(II) by phenazine, bacterialFe(II) acquisition in which the Fe(II) is obtained, and other processesidentifiable to a skilled person upon reading of the present disclosure.

The term “bacteria” as used herein as used herein refers to severalprokaryotic microbial species which include but are not limited toGram-negative and positive bacteria, such as, but not limited to,Pseudomonas, Brevibacterium, Coryneform Bacteria, NocardiaBrevibacterium linens, Brevibacterium, Burkholderia cenocepecia,Methanosarcina mazei, Mycobacterium abscessus, Pantoea agglomerans,Pectobacterium atrosepticum, Pelagio variabilis, Pseudomonasfluorescens, Streptomyces anulatus, Streptomyces cinnamonensis, andrelated species that produce phenazines to facilitate variousphysiological functions identifiable to a skilled person upon reading ofthe present disclosure. More specifically, the wording “Gram-negativebacteria” refers to bacteria that do not retain crystal violet dye inthe Gram staining protocol. In contrast, the wording “Gram-positivebacteria” refers to are those that are stained dark blue or violet byGram staining.

In some embodiments, the bacteria comprise persister cells whichtypically constitute a small portion of a culture which is tolerant tokilling by lethal doses of bactericidal antibiotics. Persister bacterialcells can be identified, for example, by exposure of logarithmic orstationary cultures of the bacteria to antibiotics using concentrationsexceeding five times the minimum inhibitory concentration for eachantibiotic. Persister numbers can be determined by plating theantibiotic-treated cultures on LB agar plates and subsequent counting ofcolony forming units representing the cell numbers which survivedantibiotic exposure. Other methods for identification of persister cellswill be known by a skilled person, and can be found, for example, inMöker et al. (“Pseudomonas aeruginosa increases formation ofmultidrug-tolerant persister cells in response to quorum-sensingsignaling molecules.” In J Bacteriol. 2010 April; 192(7):1946-55. Epub2010 Jan. 22).

In some embodiments, phenazine related pathways comprisephenazine-mediated bacterial biofilm formation, phenazine-mediated ironacquisition and phenazine mediated intracellular redox balancing ofbacteria (See Example 1-15)

In some embodiments, a phenazine related pathway comprises aphenazine-mediated signaling pathway of the bacteria. Specifically, insome embodiments, the bacteria have a motile and a sessile state and thesignaling pathway triggers a transition from the motile to the sessilestate (see Examples 6-15).

In some embodiments, one or more phenazine related pathways comprisecentral metabolic pathways of the bacteria (see Examples 11-15).

In some embodiments, the one or more phenazine related pathways comprisetransportation of phenazines in and/or out of the bacterial cell. Inother, embodiments, phenazine related pathways comprise intracellularphenazine mediated redox hemostasis of the bacteria.

In some embodiments a method and system to interfere with viability ofbacteria is described, the method comprising inactivating a phenazineand/or a phenazine-related pathway in the bacteria to reducesurvivability and/or antibiotic resistance of the bacteria.

The term “viability” as used here in refers to whether or not abacterial cell is able to maintain itself or recover its potentiality.Viable cells in the sense of the present disclosure are cells able to,or capable of recover the ability to, form colonies and biofilms on orin a solid or liquid medium. The term “medium” as used herein indicatesan environment that is suitable to support growth of microorganisms orcells. In particular, suitable medium comprise growth medium or culturemedium in a liquid or gel designed to support the bacteria in vitro, aswell as tissues and other suitable environment within a host (includinga human host) in vivo. Methods for evaluating the viability of bacteriaafter the use of the methods and systems for interference with viabilityof bacteria described herein include, but are not limited to measurementof colony forming units, cell counts such as that described by Wang etal. (J. Bacteriol. 2010, 192, 365-369), and other methods identifiableto a skilled person upon the reading of the present disclosure.

In some embodiments, inactivation of a phenazine or phenazine-relatedpathway can be performed by using small interfering RNA (siRNA)techniques to suppress the expression of proteins involved in thepathway.

The term “small interfering RNA (siRNA)” as used herein refers to aclass of double stranded RNA molecules, typically of 20-25 nucleotidesin length, that play varying roles in biology including suppression ofgene expression and are identifiable by a skilled person. AppropriatesiRNA sequences can be selected based on the sequence of the gene thatis to be suppressed, for example, by use of a sequence designer such asInvivoGen's online siRNA designer (available at the filing date at thewww page sirnawizard.com), or by other methods known to a skilledperson. The siRNA oligonucleotide sequences can then be introduced intothe bacteria, for example, by culturing them with the bacteria ofinterest to suppress the expression of the gene of interest (as seen,for example, in Yanagihara et al., J. Antimicrob. Chemotherapy 2005, 57,122-126). Additional methods for interfering with gene expression by RNAinterference are identifiable by a skilled person (see, for example,Lehner et al. “How to use RNA interference” Briefings in FunctionalGenomics and Proteomics, 2004, 3, 68-83) and can include, but are notlimited to use of micro RNA (miRNA), small nucleolar RNA (snoRNA), andothers identifiable to a skilled person.

In some embodiments, inactivating a phenazine or phenazine-relatedpathway can be performed by inhibiting synthesis of the phenazine in thebacteria (Example 1). An example of the pathways for phenazinebiosynthesis in bacteria is illustrated in Mentel et al. (ChemBioChem2009, 10, 2295-2304) and reported in the scheme herein below.

In some embodiments, inhibiting synthesis of a phenazine in the bacteriacan be performed by inactivating phenazine biosynthetic genes (see, forexample, Example 1), e.g. by blocking transcription and/or blockingtranslation of said genes. Exemplary phenazine biosynthetic genesinclude, but are not limited to, genes coding for the phenazinebiosynthesis proteins such as PhzA, PhzB, PhzC, PhzD, PhzE, PhzF, PhzF1,PhzF2, PhzG, PhzG1, PhzG2, PhzM, PhzH, PhzS and other proteins andhomologs identifiable to a skilled person, such as those indicated inMentel et al (ChemBioChem 2009, 10, 2295-2304) and Dietrich et al.(Molecular Microbiology 2006, 61, 1308-1321). In some of thoseembodiments, inactivating can be performed for example by blockingtranscription through the siRNA techniques described herein.

In particular, in some embodiments, inhibiting synthesis of thephenazine can be performed by inactivating the relevant biosyntheticpathway, e.g. by inhibiting specific enzymes involved in the synthesisof phenazine from the relevant starting compound (e.g.erythrose-4-phosphate and phosphoenol pyruvate) such as PhzF, anessential diaminopimelate epimerase-like enzyme (Blankenfeldt, PNAS2004, 101, 16431-16436) which can be inhibited by an aziridinodiaminopimelate (see Pillai et al. Proc. Natl, Acad. Sci. 2006, 103,8668-8673). Other enzymes involved in phenazine biosynthesis that can beinhibited in accordance with the present disclosure and their respectiveinhibitors are identifiable by a skilled person upon reading of thepresent disclosure, and include, but not limited to, inhibition of PhzG,a flavin-dependent oxidase (see Parsons et al. Acta CrystallographicaSection D, 2004, 60, 2110-2113) byN⁵,N¹⁰-methylene-5,6,7,8-tetrahydrofolate, an inhibitor of theflavin-dependent oxidase FDTS (see Wang et al. FEBS J. 2009, 276,2801-2810), and inhibition of PhzE by divalent ions such as, but notlimited to, Zn²⁺, Mn²⁺, and Ni²⁺ (as seen, for example, in Li et al. J.Biol. Chem. 2011, 286, 18213).

In some embodiments, inactivating a phenazine or phenazine-relatedpathway is performed by inactivating a phenazine-mediated signalingpathway. In some embodiments, inactivating a phenazine-mediatedsignaling pathway is performed by inactivating one or more signalingmolecules in the phenazine-mediated signaling pathway.

In particular, in some embodiments, the one or more signaling moleculesare in the form of a protein. In other embodiments, the signalingmolecules are direct or indirect effectors of phenazines in the pathway.Signaling molecule can be identified by a skilled person, and include,but are not limited to, signaling molecules mentioned in Dietrich et al.(Molecular Microbiology 2006, 61, 1308-1321). Specifically, in someembodiments, the one or more signaling molecules comprise acylhomoserine lactones such as, for example 3-oxo-dodecanoyl-homoserinelactone (3-oxo-C₁₂-HSL) and butanoylhomoserine lactone (C₄-HSL), andquinolones such as, for example, the pseudomonas quinolone signal (PQS).(see, for example, Example 17 and Dietrich et al. Molecular Microbiology2006, 61, 1308-1321).

In some embodiments, inactivating one or more signaling molecules isperformed by inhibiting expression of one or more genes in the bacteriacoding for signaling molecules in the pathway. In an exemplaryembodiment, the gene suppressed is the pqsH gene, which codes for theprotein PqsH that synthesizes PQS, and it is suppressed as describedabove by using the siRNA techniques herein described (see, for example,Example 17 and Dietrich et al. Molecular Microbiology 2006, 61,1308-1321).

In particular, in some embodiments, inactivating a phenazine orphenazine-related pathway can be performed by inactivating one or moreproteins that recognize signaling molecules. In some of thoseembodiments, the one or more proteins comprise proteins that recognizeacyl homoserine lactones such as 3-oxo-C₁₂-HSL and C₄-HSL. Examples ofproteins that recognize acyl homoserine lactones include, but are notlimited to, LasR and RhlR. In an exemplary embodiment, LasR and RhlR areinactivated by suppressing the genes lasR and rhlR using the siRNAtechniques herein described. Additional methods of inactivating theproteins such as LasR, RhlR, and PhzA1-G1 can be identified by a skilledperson upon reading of the present disclosure (see, for example, Example17 and Dietrich et al. Molecular Microbiology 2006, 61, 1308-1321).

Additional signaling molecules suitable in the methods and systemsherein described, are identifiable by a person skilled in the art uponreading of the present disclosure. In an exemplary embodiment,identification of signaling molecule can be performed by generatingmutant bacterial strains with defective phenazine biosynthetic pathways,for example, by transposing the relevant DNA sequences and identifyingfrom the gene sequences the related proteins involved in phenazinebiosynthesis e.g. by using the techniques of Gallagher et al. (J.Bacteriol. 2002, 184, 6472-6480; incorporated herein by reference in itsentirety).

In some embodiments, inactivating a phenazine or phenazine-relatedpathway can be performed by interfering with quorum sensing of bacteriaand in particular with the proteins and other molecules involved in therelated pathway, which is more particularly associated with phenazinebiosynthesis (see Example 17).

The term “quorum sensing” as used herein refers to a system of stimulusand response correlated to bacterial population density typical ofcertain bacteria identifiable by a skilled person. Bacteria havingquorum sensing, use quorum sensing to coordinate gene expressionaccording to the density of their local population. Bacteria that usequorum sensing constantly produce and secrete certain signalingmolecules (called autoinducers or pheromones). These bacteria also havea receptor that can specifically detect the signaling molecule(inducer). When the inducer binds the receptor, it activatestranscription of certain genes, including those for inducer synthesis.There is a low likelihood of a bacterium detecting its own secretedinducer. Thus, in order for gene transcription to be activated, the cellmust encounter signaling molecules secreted by other cells in itsenvironment. When only a few other bacteria of the same kind are in thevicinity, diffusion reduces the concentration of the inducer in thesurrounding medium to almost zero, so the bacteria produce littleinducer. However, as the population grows, the concentration of theinducer passes a threshold, causing more inducer to be synthesized. Thisforms a positive feedback loop, and the receptor becomes fullyactivated. Activation of the receptor induces the upregulation of otherspecific genes, causing all of the cells to begin transcription atapproximately the same time. A model of a bacterial quorum sensingnetwork and its association with phenazine synthesis can be seen, forexample, in Dietrich et al. (Molecular Microbiology 2006, 61, 1308-1321)and is illustrated in FIG. 19.

In some embodiments, interfering with the quorum sensing of the bacteriacan be performed by inhibiting transcription and/or translation of thephenazine biosynthetic genes. In an exemplary embodiment, interferingwith quorum sensing can be performed using siRNA oligonucleotidescorresponding to the gene sequences of pqsH (as seen, for example, inGallagher et al. J. Bacteriol. 2002, 184, 6472-6480). In particular, insome embodiments siRNA oligonucleotides corresponding to the genesequences of pqsH can be added to the bacteria and be incorporated intothe bacteria using methods and techniques identifiable by a skilledperson upon reading of the present disclosure. In some embodiments, thesiRNA oligonucleotides can then interfere with the expression of pqsHwhich in turn can result in the inhibited biosynthesis of Pseudomonasquinolone signal (PQS) and subsequently results in reduced phenazinebiosynthesis as seen, for example, in Dietrich et al. (MolecularMicrobiology 2006, 61, 1308-1321). Additional genes to target by siRNAmethods described herein include, but are not limited to, other genesthat code for proteins involved in quorum sensing such as, but notlimited to, LasR, Rhll, RhlR, MvfR and others, as indicated in, forexample, Dietrich et al. (Molecular Microbiology 2006, 61, 1308-1321)(see, for example, Example 17).

In another exemplary embodiment, bacterial strains can be grown in thepresence of quorum-sensing inhibitors such as, tobramycin (see, forexample, Babic “Tobramycin at subinhibitory concentration inhibits theRhll/R quorum sensing system in a Pseudomonas aeruginosa environmentalisolate” Infectious Diseases 2010, 10:148 and Garske et al. Pathology2004, 36, 571-575) or furanone C-30 (Hentzer et al. EMBO J. 2003 Aug. 1;22(15): 3803-3815), or others identifiable to a skilled person.

In some embodiments, inactivating a phenazine related pathway can beperformed by inactivating intracellular phenazine mediated redoxhemostasis of the bacteria is performed by inhibiting phenazine-mediatedelectron shuttling of the bacteria. In a non-limiting example, thephenazine-mediated electron shuttling of the bacteria is inhibited byinhibiting the biosynthesis of phenazines as described above. (SeeExamples 6-15).

In some embodiments, inactivating a phenazine or phenazine relatedpathway can be performed by reducing the amount of phenazine inbacteria, e.g. by any of techniques and approaches herein describedperformed to reduce rather than minimize the amount of phenazinesynthesized by bacteria (see, for example, Example 1). The amount ofphenazine in the bacteria before and after reduction of the quantity ofphenazine according to the methods described herein can be measured bymethods identifiable to a skilled person upon reading of the presentdisclosure. For example, the quantity of phenazine in bacterial culturebefore and after reduction of the quantity of phenazine can be measuredby directly loading the filtrate of the culture onto a HPLC column andanalyzing the filtrate as done by Dietrich et al. (MolecularMicrobiology 2006, 61, 1308-1321). Additional quantification techniquescan be identified by a skilled person and can include, for example,using time-lapsed spectral multiphoton fluorescence microscopy ofSullivan et al., (ACS Chemical Biology 2011, 6, 893-899) to monitorphenazine concentrations within bacterial cells in vivo both before andafter reduction of the phenazine levels.

In some embodiments, reducing the amount of phenazine in bacteria can beperformed by enhancing phenazine degradation endogenously and/orexogenously.

In particular, in some embodiments, enhancing phenazine degradation canbe performed by expressing and/or delivering a protein that degradesphenazines. In an exemplary embodiment, a DNA sequence of aphenazine-degrading protein can be delivered by introduction of the DNAsequence into a bacterium via a vector (e.g. viral vector), or othertechniques identifiable by a skilled person upon reading of the presentdisclosure, and the DNA sequence expressed in the bacteria to producethe phenazine-degrading protein. In another embodiment,phenazine-degrading proteins can be expressed in other bacteria and thenisolated and purified to afford phenazine-degrading proteins suitablefor extracellular degradation of phenazine (see for example, Examples 15and 18).

In some embodiments, inactivating a phenazine or phenazine-relatedpathway is performed by providing the bacteria with one or morephenazine-degrading enzymes. Bacteria that degrade phenazines have beenisolated and identified (see Examples 15 and 18).

In some embodiments, enzymes degrading a phenazine can be identified byfirst identifying a bacterium capable of phenazine degradation. Theidentification can be performed, for example, by constructing abacterial “enrichment culture” by defining a minimal growth medium wherea phenazine (PCA, PYO, and additional phenazines identifiable by askilled person) is provided as either (or both) the sole source ofcarbon or nitrogen. If growth is observed after many rounds of serialdilutions, phenazine-degraders can be isolated by plating the enrichmentculture on an agar plate with the same medium composition. Singlecolonies are picked, and streaked to fresh plates, and visually checkedfor purity. Once pure, the 16S rDNA is sequenced and the organism can bephenotypically characterized. Other methods for identifying a bacteriumcapable of phenazine degradation would be identifiable to a skilledperson upon reading of the present disclosure. Once a bacterium capableof degrading phenazine is identified, one or more particular enzymesresponsible for phenazine degradation in the bacterium can beidentified, for example, by biochemical approach and/or geneticapproaches (see, for example, Examples 15 and 18).

In an exemplary embodiment, the bacteria capable of producingphenazine-degrading enzymes are Sphingomonas sp. DP58 (see Yang et al.Current Microbiology 2007, 55, 284-287 and Chen et al. Biodegradation2008, 19, 659-667).

In particular, a biochemical approach can comprise performing anactivity assay, for example based on absorption or fluorescence aphenazine over time and a subsequent purifying of cell fractions topromote a disappearance of phenazine.

A genetic approach can comprise employing transposition mutagenesis tomake a collection of random mutants and screening them for an inabilityto grow on a minimal medium plus the phenazine, as described, forexample, in Gallagher et al. (J. Bacteriol. 2002, 184, 6472-6480).

Further to these methods, once an enzyme is identified, the specificityof the enzyme can be altered using directed evolution, such thatfollowing directed evolution the enzyme can recognize either a specificphenazine, a broader range of phenazines, and/or to improve efficiencyof the enzyme. In an exemplary embodiment, the genetic sequencecorresponding to the phenazine degrading enzyme can be randomly mutatedusing error-prone PCR or another technique identifiable to the skilledperson to produce a library of mutated genetic sequences. The proteinsexpressed by the mutant sequences can be screen for phenazine degradingactivity against specific or broad ranges of phenazines, for example, bythe spectrophotometric measurement of phenazine levels over time. Theproteins thus identified to be able to degrade a specific phenazine orbroad range of phenazines can be synthesized, for example, in abacterium using recombinant DNA techniques known to the skilled person(see, for example, Examples 15 and 18).

In other embodiments, reducing the amount of phenazine in bacteria isperformed by modifying the phenazines (e.g. chemically) to interfere andin particular minimize phenazine uptake by the bacteria. Exemplarymodifications include, but are not limited to, direct chemicalmodification of the phenazines such as, epoxidation of reactive doublebonds, alkylation of nucleophilic groups, acylation of nucleophilicgroups, electrophilic aromatic substitution of one or more hydrogenatoms on the phenazines, and other chemical modifications identifiableto a skilled person using reaction conditions identifiable to a skilledperson.

In some embodiments, inactivating a phenazine or phenazine relatedpathway is performed by inhibiting transportation of phenazines inand/or out of a bacterial cell. In particular, in some embodiments,inhibiting transportation of phenazines in and/or out of a bacterialcell can be performed by blocking one or more phenazine exporters ofbacteria. Suitable bacterial phenazine exporter inhibitors include, butare not limited to, Phe-Arg-3-naphthylamide (PA3N),Pro-D-hPhe-3-aminoquinolone (MC-04,124), and MexAB-OprM-specific EPID13-9001, and others identifiable to a skilled person upon reading ofthe present disclosure (additional inhibitors can be found, for example,in Hirakata et al., 2009, Int. J. Antimicrob. Agents, 34:343-346, andAskoura, Libyan J Med, 2011, 6, 5870). In an exemplary embodiment,bacteria can be inoculated onto an agar plate into which has beenincorporated a phenazine exporter inhibitor such as done, for example,by Saenz et al. (J. Antimicrob. Chemotherapy 2004, 544) and the efficacyof inhibition determined, for example, by the method of Fritsche et al(Antimicrobial Agents and Chemotherapy, 2005, 49, 1468-1476).

In some embodiments, the one or more phenazine exporters of bacteriacomprise RND efflux pumps of the mexGHI-opmD variety. (see Example 17).In an exemplary embodiment, the activity of the efflux pump mexGHI-opmDcan be inhibited by, for example, preventing its expression withappropriate siRNA oligonucleotides as described above.

In some embodiments, the bacterium is Pseudomonas aeruginosa, and theone or more phenazine export proteins of bacteria can be encoded bygenes PA4205, PA4206, PA4207 and/or PA4208, and the phenazineexportation is disrupted by deleting of these genes or by decreasingtheir expression. In an exemplary embodiment, gene sequences can bedeleted by generating the appropriate integration cassette connected tothe upstream and downstream regions flanking the genes to be deleted andtransforming the generated recombinant DNA into the bacterial genome,for example, in a liquid culture, as done for example by Berardinis etal. (“A complete collection of single-gene deletion mutants ofAcinetobacter baylyi ADP” Molecular Systems Biology 2008 4: 174) andDietrich et al. (Molecular Microbiology 2006, 61, 1308-1321). (See, forexample, Example 1)

In some embodiments, inhibiting transportation of phenazines in and/orout of a bacterial cell is performed by blocking a protein involved inmodifying phenazines to be recognized by a phenazine exporter ofbacteria (see Example 17). Proteins involved in modifying phenazines tobe recognized by a phenazine exporter include, but are not limited to,the phenazine-decorating PhzM, PhzS, and PhzH indicated by Dietrich etal. (Molecular Microbiology 2006, 61, 1308-1321). These proteins, whilerequired for addition of functional groups, are not required for proteinexport. Methods for blocking these proteins include, but are not limitedto, the use of inhibitors as with PhzF above, and the use of siRNAoligonucleotides to suppress their expression as described above.

In some embodiments, the bacterium is Pseudomonas aeruginosa, and thephenazine related pathway is redox sensing of bacteria and the proteininvolved is the protein encoded by gene PA2274 (see Example 17). Theexpression of such a gene can be suppressed by use of the siRNAoligonucleotides as described above. Alternatively, as the coded enzymeis a flavin-dependent monoxygenase, it is expected to be inhibited byN⁵,N¹⁰-methylene-5,6,7,8-tetrahydrofolate in substantially the samemanner as PhzG above.

In some embodiments, inhibiting transportation of phenazines in and/orout of a bacterial cell can be performed by blocking one or more MFStransporters involved in phenazine import/export of bacteria hereindescribed.

In some embodiments, the bacterium is Pseudomonas aeruginosa, and theone or more MFS transporters involved in phenazine import/export ofbacteria can be encoded by the genes PA3718 and/or PA4233. In anexemplary embodiment, the MFS transporters encoded by genes PA3718and/or PA4233 can be blocked by suppression of their expression by theirgenes using the siRNA techniques described herein. In an alternative,non-limiting example, the MFS transporters can be blocked using theinhibitor Phe-Arg-β-naphthylamide and the methods of Saenz et al. (J.Antimicrob. Chemotherapy 2004, 544) and Fritsche et al. (AntimicrobialAgents and Chemotherapy, 2005, 49, 1468-1476). Additional inhibitorwould be identifiable to a skilled person and can include, for example,inhibitors described by Vecchione et al. (Antimicrob. Agents andChemotherapy, 2009, 53, 4673-4677) (see, for example, Example 1 andDietrich et al. Molecular Microbiology 2006, 61, 1308-1321).

In some embodiments, inactivating a phenazine or phenazine relatedpathway is performed by converting at least a portion of the phenazineof the bacteria to an inactive form. Specifically, in some embodiments,converting at least a portion of the phenazine to an inactive form isperformed by inhibiting intracellular reduction and/or extracellularoxidation of phenazines of the bacteria. In an exemplary embodiment,inhibition of intracellular reduction of phenazines can be performed bygrowing bacterial cultures in a bacterial growth medium, for example,containing compounds with structures analogous to phenazines capable ofinhibiting the intracellular reduction of phenazines including, but notlimited to, methylene blue, paraquat, and others identifiable to askilled person. In another exemplary embodiment, extracellular oxidationof phenazine can be inhibited by removing a oxidant, such as Fe(III),(see, for example, Hernandez et al. Applied and EnvironmentalMicrobiology 2004, 70, 921-928) by growing bacterial cultures in abacterial growth medium containing an Fe(III) chelator such as, forexample, conalbumin as indicated, for example, by Wang et al. (J.Bacteriol. 2011, 193, 3606-3617). Additional methods of inhibitingintracellular reduction and/or extracellular oxidation of phenazines ofthe bacteria will be identifiable to a skilled person upon reading ofthe present disclosure.

Therefore, inactivating phenazines can be performed in some embodiments,by interfering with phenazine uptake and/or intracellular processing ofthe bacteria (see Example 14). Thus, in some embodiments, converting atleast a portion of the phenazine to an inactive form can be performed bymodifying phenazines (e.g. chemically) to interfere with phenazineuptake and/or intracellular processing of bacteria.

In some embodiments, inactivating a phenazine or phenazine-relatedpathway comprises impairing phenazine-mediated bacterial biofilmdevelopment in the bacteria.

As used herein the term “biofilm” indicates an aggregate ofmicroorganisms in which cells adhere to each other on a surface. Theseadherent cells are frequently embedded within a self-produced matrix ofextracellular polymeric substance (EPS). Biofilms can form on living ornon-living surfaces and can be prevalent in natural, industrial andhospital settings. The microbial cells growing in a biofilm arephysiologically distinct from planktonic cells of the same organism,which, by contrast, are single-cells that can float or swim in a liquidmedium. Formation of a biofilm begins with the attachment offree-floating microorganisms to a surface. These first colonists adhereto the surface initially through weak, reversible adhesion via van derWaals forces. If the colonists are not immediately separated from thesurface, they can anchor themselves more permanently using cell adhesionstructures such as pili. When the biofilm growth is balanced with thatof biofilm dispersion, the biofilm is considered “mature.” Methods toquantify and measure biofilms will be known to a skilled person and caninclude, for example, the COMSTAT method of Heydorn et al. (Microbiology2000, 146, 2395-2407).

In some embodiments, the phenazine-mediated bacterial biofilmdevelopment comprises phenazine-mediated iron acquisition of bacteria.Iron has been shown to be involved as a signal in bacterial biofilmformation (see, for example, Banin et al. PNAS, 2005, 102, 11076-11081).Phenazines have been shown to mediate iron acquisition in bacterialbiofilm development, for example, by reduction of insoluble Fe(III) tomore soluble Fe(II) (See, for example, Wang et al. J. Bacteriol. 2011,193, 3606-3617, and Examples 1-6).

In some embodiments, inactivating a phenazine related pathway comprisesinactivating phenazine-mediated iron acquisition of bacteria. In anexemplary embodiment, the inactivation of the phenazine-mediated ironacquisition is performed by reducing the amount of phenazine inbacteria, e.g. by any of techniques and approaches herein described toreduce the amount of phenazine available to reduce Fe(III) to Fe(II)prior to acquisition by the bacteria (see, for example, Examples 1-6).

In other embodiments, the inactivation of the phenazine-mediated ironacquisition is performed by subtracting iron from the medium hosting thebacteria.

The term “subtraction” as used herein with reference to iron refers tothe at least partial removal of iron in any of its oxidation states froma bacteria or its local environment such that the subtracted iron is notable to be acquired or otherwise used by the bacteria. Exemplary ironsubtraction can be performed by ion exchange, precipitation of the iron,sequestration of the iron, and other approaches and techniquesidentifiable to the skilled person upon reading of the presentdisclosure.

In some embodiments, the subtracting is performed by use of ironchelators. The term “chelator” as used herein refers to a moleculecapable of binding a metal ion (e.g. iron) by forming multiple bonds tothe metal. Chelators can be biological molecules (such as, hemoglobin,transferrin, lactoferrin, conalbumin and ferritin; or siderophores suchas deferoxamine, deferiprone, deferasirox, 2,2-dipyridyl,1,10-phenanthroline, Ferrozine® and Enterobactin; or others identifiableto a skilled person) or organic chelators (such as EDTA,diethylenetriamine, ethylenediamine,N,N′,N″-tris(2-pyridylmethyl)-1,3,5-cis,cis-triaminocyclohexane(tachpyr), and others identifiable to a skilled person).

In other embodiments, inactivating the pathway of phenazine-mediatediron acquisition by bacteria is performed by inhibiting Fe(II)acquisition by bacteria. In particular, in some embodiments, inhibitingFe(II) acquisition by bacteria can be performed by inhibitingcytoplasmic membrane Fe(II) transporter of bacteria. In someembodiments, the Fe(II) transporter is the cytoplasmic membrane proteinFeoB or a homologues protein thereof identifiable to a skilled person(such as, but not limited to, FeoB1 and FeoB2; see, for example, Dasperet al. The Journal of Biological Chemistry 2005, 280, 28095-28102, andExample 3 below). Other suitable cytoplasmic membrane Fe(II)transporters can be identified by a skilled person, and can includethose in bacteria which substantially do not grow anaerobically onFe(II). In an exemplary embodiment, the inhibition of FeoB can beperformed by suppressing FeoB expression using the siRNA techniquesdescribed above. In an additional non-limiting example, due to FeoBbeing a transporter of divalent iron, FeoB can be inhibited byinhibitors of other divalent metal transporters such as, but not limitedto, NSC306711 and NSC75600 and others identifiable to a skilled person(see, for example, Buckett et al. Am. J. Physiol. Gastrointest. LiverPhysiol. 2009, 296, G798-G804).

In other embodiments, inactivating the pathway of phenazine-mediatediron acquisition can be performed by exposing bacteria to an Fe(II)chelator. Specifically, in some embodiments, the Fe(II) chelator is inthe form of a protein and/or a chemical compound. In an embodiment, theactivation of the Fe(II) chelator can be performed by adding the Fe(II)chelator to a bacterial culture in a way similar to the use ofconalbumin to chelate Fe(III) described herein (see, for example,Example 3).

In some embodiments, the Fe(II) chelator is Ferrozine®, and activatingof the Fe(II) chelator can be performed by delivering Ferrozine® into,for example, the mucus environment of bacteria (see, for example,Examples 21-22).

In some embodiments the Fe(II) chelator is in the form of an aerosol andcan thus be delivered topically, e.g. directly into the lungs of apatient. Methods to deliver the Fe(II) chelator into the lungs of apatient can be identified by a skilled person using, for example, themethods of Corkery (“Inhalable Drugs for Systemic Therapy” RespiratoryCare 2000, 45, 931-835) (see, for example, Examples 19-20).

In other embodiments, the Fe(II) chelator is a host protein, andactivating a Fe(II) chelator comprises regulating of one or more hostgenes encoding a host Fe(II) chelator. Fe(II) chelating host proteinsare identifiable to a skilled person can include, but not be limited to,apoferritin and methods for regulating the host genes encoding the hostFe(II) chelators can be identified by a skilled person and can include,but not be limited to, use of the siRNA techniques described above.

In some embodiments, inactivating the pathway of phenazine-mediated ironacquisition of bacteria can be performed by inhibitingphenazine-mediated Fe (III) reduction to Fe(II) (see, for example,Examples 1-4). In an exemplary embodiment, the phenazine-mediatedFe(III) reduction to Fe(II) can be inhibited by reducing amount ofFe(III) available to a bacterial culture by a suitable addition of ironchelator such as conalbumin, to the bacterial culture. As seen inExample 3 and, for example, and in Wang et al. (J. Bacteriol. 2011, 193,3606-3617) the biofilm development was impaired when in presence ofconalbumin. Other iron-binding molecules usable in this method would beidentifiable to skilled person and can include, for example, EDTA,desferrioxamine, hemoglobin, transferrin, lactoferrin, and ferritin.

In some embodiments, inactivating the pathway of phenazine-mediated ironacquisition can be performed by activating a Fe(III) chelator in thebacteria. Specifically, in some embodiments, the Fe(III) chelator is inthe form of a protein and/or a chemical compound (see Examples 19-22).In exemplary embodiment, the activation of the Fe(III) chelator can beperformed by adding the Fe(III) chelator to a bacterial culture the in away similar to the use of conalbumin to chelate Fe(III) to inhibit itsreduction to Fe(II) described above in reference to Example 3 below. Inanother non-limiting example, a DNA sequence of a Fe(III) chelatingprotein delivered by introduction of the DNA sequence into a bacteriavia a virus, or another technique identifiable to a skilled person uponreading of the present disclosure, and the DNA sequence expressed in thebacteria to produce the Fe(III) chelating protein. Appropriate Fe(III)chelating proteins can include, but are not limited to, hemoglobin,transferrin, lactoferrin, and ferritin.

In some embodiments, the Fe(III) chelator is conalbumin, and activatinga Fe(III) chelator can be performed by delivering conalbumin into themucus environment of bacteria (see, for example, Examples 3 and 21-22).

In some embodiments the Fe(III) chelator is in the form of an aerosoland can thus be delivered directly into the lungs of a patient. Methodsto deliver the Fe(III) chelator into the lungs of a patient can beidentified by a skilled person using, for example, the methods ofCorkery (“Inhalable Drugs for Systemic Therapy” Respiratory Care 2000,45, 931-835) (see, for example, Examples 19-20).

In other embodiments, the Fe(III) chelator is a host protein, andactivating a Fe (III) chelator comprises regulating of one or more hostgenes encoding a host Fe(III) chelator. Fe(III) chelating host proteinsare identifiable to a skilled person can include, but not be limited to,hemoglobin, transferrin, lactoferrin, conalbumin, and ferritin, andmethods for regulating the host genes encoding the host Fe(III)chelators can be identified by a skilled person and can include, but notbe limited to, use of the siRNA techniques described above (see, forexample, Example 3 and 21-22).

In some embodiments herein described, iron chelation can be used toinhibit pathogenic microbial biofilms in vitro and in vivo.

In some embodiments, Fe(II) and Fe(III) chelators can be activated incombination to substantially minimize and/or disrupt biofilm growth asexemplified in Examples 21-22. In these embodiments, Fe(II) and Fe(III)chelators can act synergistically to substantially prevent and/ordisrupt biofilm growth as also exemplified in Examples 21-22.

In some embodiments the Fe(II) chelator to be used in combination withan Fe(III) chelator is Ferrozine®. In some embodiments the Fe(III)chelator to be used in combination with an Fe(II) chelator isconalbumin. In some embodiments, the Fe(II) chelator and Fe(III)chelator administered in combination can be Ferrozine® and conalbumin(see, for example, Examples 21-22).

In particular, in some embodiments, the combination of activation ofFe(II) and Fe(III) chelators in combination can be used to target maturebiofilms. Mature biofilms are of significance, for example, becauseincreased resistance to antibiotics (see, for example, Ito et al.Applied and Environmental Microbiology 2009, 75, 4093-4100 and Example22).

Thus, in some embodiments, a method for interfering with viability ofbacteria comprises activating a combination of Fe(II) and Fe(III)chelators to substantially prevent and/or disrupt biofilm growth. Inthese embodiments, Fe(II) and Fe(III) chelators can act synergisticallyto substantially prevent and/or disrupt biofilm growth and can be usedto target mature biofilms. (See, for example, Example 22).

The ability of the combination of Fe(II) and Fe(III) chelators insubstantially preventing and/or disrupting biofilm growth can be due tothe appreciable levels of ferrous iron [Fe(II)] which can exist in themajority of CF lung which can compromise Fe(III) chelation therapy underhypoxic or anoxic conditions. Such appreciable levels of Fe(II) can bedue to localized hypoxic microenvironments exist which can stabilizeFe(II) (see, for example, Examples 19-22).

Thus, in some embodiments, a treatment for cystic fibrosis (CF) patientscomprises administering Fe(II) and Fe(III) chelators in combination tosubstantially prevent and/or disrupt biofilm growth. (See, for example,Examples 19-22).

In some embodiments, compositions for substantially preventing and/orreducing biofilms are described. The composition comprises one or moreagents able to inactivate a phenazine and/or a phenazine related pathwayin the bacteria to reduce survivability of bacteria. (See, for example,Examples 1-5 and 19-22).

In some embodiments the composition comprises an Fe(II) chelator and anFe(III) chelator. In some embodiments, the Fe(II) chelator is Ferrozine®and is comprised in the composition in an amount ranging between about10 and about 1000 μM. In some embodiments, the Fe(III) chelator isconalbumin and is comprised in the composition in an amount rangingbetween about 10-1000 μM (see, for example, Examples 1-5 and 21-22).

In some embodiments, the composition comprises Ferrozine® in an amountranging between about 10 and about 1000 μM and conalbumin Ferrozine® inan amount ranging between 10-1000 μM (see, for example, Examples 1-5 and21-22).

In some embodiments, the composition comprises Ferrozine® in an amountof approximately 200 μM (see, for example, Examples 1-5 and 21-22).

In some embodiments, the composition comprises conalbumin in an amountof approximately 100 μM (see, for example, Examples 1-5 and 21-22).

In some embodiments, the composition comprising administering acombination of the Fe(II) chelator and the Fe(III) chelator to reducebiofilm accumulation by greater than approximately 20%. In someembodiments, composition comprising the combination of the Fe(II)chelator and the Fe(III) chelator reduces biofilm accumulation bygreater than approximately 50% (see, for example, Examples 21-22).

In some embodiments, a method and system to interfere with the viabilityof bacteria is described, the method comprising the chelation of Fe(II)alone/or Fe(III) to reduce survivability and/or antibiotic resistance ofthe bacteria.

In some embodiments, Fe(II) and Fe(III) chelators can be activated incombination to substantially prevent and/or disrupt biofilm growth asexemplified in Examples 21-22. In these embodiments, Fe(II) and Fe(III)chelators can act synergistically to substantially prevent and/ordisrupt biofilm growth as also exemplified in Examples 21-22.

In some embodiments the Fe(II) chelator to be used in combination withan Fe(III) chelator is Ferrozine®. In some embodiments the Fe(III)chelator to be used in combination with an Fe(II) chelator isconalbumin. In some embodiments, the Fe(II) chelator and Fe(III)chelator administered in combination can be Ferrozine® and conalbumin(see, for example, Examples 21-22).

In particular, in some embodiments, the combination of activation ofFe(II) and Fe(III) chelators in combination can be used to target maturebiofilms. Mature biofilms are of significance, for example, becauseincreased resistance to antibiotics (see, for example, Ito et al.Applied and Environmental Microbiology 2009, 75, 4093-4100).

Thus, in some embodiments, a method for interfering with viability ofbacteria comprises activating a combination of Fe(II) and Fe(III)chelators to substantially prevent and/or disrupt biofilm growth. Inthese embodiments, Fe(III) and Fe(II) chelators can act synergisticallyto substantially prevent and/or disrupt biofilm growth and can be usedto target mature biofilms. (See Example 22).

The ability of the combination of Fe(III) and Fe(II) chelators insubstantially preventing and/or disrupting biofilm growth can be due tothe appreciable levels of ferrous iron [Fe(II)] which can exist in themajority of CF lung which can compromise Fe(III) chelation therapy underhypoxic conditions. Such appreciable levels of Fe(II) can be due tolocalized hypoxic microenvironments exist which can stabilize Fe(II)(see, for example, Examples 19-22).

Thus, in some embodiments, a treatment for cystic fibrosis (CF) patientscomprises administering Fe(III) and Fe(II) chelators in combination tosubstantially prevent and/or disrupt biofilm growth. (See, for example,Examples 19-22).

In some embodiments, compositions for substantially preventing and/orreducing biofilms are described. The composition comprises one or moreagents able to chelate Fe(II) and/or Fe(III) to reduce survivability ofbacteria. (See, for example, Examples 19-22).

In some embodiments the composition comprises an Fe(II) chelator and anFe(III) chelator. In some embodiments, the Fe(II) chelator is Ferrozine®and is comprised in the composition in an amount ranging between about10 and about 1000 μM. In some embodiments, the Fe(III) chelator isconalbumin and is comprised in the composition in an amount rangingbetween about 10 and about 1000 μM (see, for example, Examples 1-5 and21-22).

In some embodiments, the composition comprises Ferrozine® in an amountranging between about 10 and about 1000 μM and conalbumin Ferrozine® inan amount ranging between 10-1000 μM (see, for example, Examples 1-5 and21-22).

In some embodiments, the composition comprises Ferrozine® in an amountof approximately 200 μM (see, for example, Examples 1-5 and 21-22).

In some embodiments, the composition comprises conalbumin in an amountof approximately 100 μM (see, for example, Examples 1-5 and 21-22).

In some embodiments, the composition comprising a combination of theFe(II) chelator and the Fe(III) chelator reduces biofilm accumulation bygreater than approximately 20%. In some embodiments, compositioncomprising the combination of the Fe(II) chelator and the Fe(III)chelator reduces biofilm accumulation by greater than approximately 50%(see, for example, Examples 21-22).

In some embodiments, the methods mentioned above can further comprisedegrading phenazines in vivo and/or in vitro using methods and systemsherein described.

Further, in some embodiments, a method and system for treating and/orpreventing a bacterial infection in an individual is described. Themethod comprises administering an effective amount of one or more agentsable to selectively inactivate phenazine and/or a phenazine relatedpathway in the bacteria, in particular in combination with an antibioticand/or other antimicrobial. The system comprises one or more agents ableto specifically inactivate a phenazine and/or a phenazine relatedpathway in the bacteria and an antibiotic and/or other antimicrobial(see, for example, Examples 19-22).

Further, in other embodiments, a method and system for treating and/orpreventing a bacterial infection in an individual is described. Themethod comprises administering an effective amount of one or more agentsable to selectively chelate Fe(II) and/or Fe(III), in particular incombination with an antibiotic and/or other antimicrobial. The systemcomprises one or more agents able to specifically chelate Fe(II) and/orFe(III) and an antibiotic and/or other antimicrobial (see, for example,Examples 1-5 and 19-22).

In particular, in some embodiments, a method for treating and/orpreventing bacterial infection associated with cystic fibrosis isdescribed. The method comprises administering a therapeuticallyeffective amount of a combination of Fe(III) and Fe(II) chelators to anindividual (see, for example, Examples 19-22).

In some embodiments the administering can be performed by way of anaerosol comprising the Fe(III) and Fe(II) chelators, however other formsof administration, identifiable by a skilled person, can be used.

In some embodiment, the administering of the Fe(III) and Fe(II)chelators substantially prevents and/or disrupts biofilm growth in thelungs of an individual infected with the bacteria, such as a CF patient(see, for example, Examples 19-22).

In some embodiments, Fe(III) and Fe(II) chelators can actsynergistically to substantially prevent and/or disrupt biofilm growthand can be used to target mature biofilms (see, for example, Examples19-22).

In some embodiments, the Fe(II) chelator is Ferrozine® and atherapeutically effective amount ranges from approximately 10 toapproximately 1000 μM. More particularly, in some embodiments, thetherapeutically effective amount of Ferrozine® is approximately 200 μM(see, for example, Examples 19-22).

In some embodiments, the Fe(III) chelator is conalbumin and atherapeutically effective amount ranges from approximately 10 toapproximately 1000 μM. More particularly, in some embodiments, thetherapeutically effective amount of conalbumin is approximately 100 μM(see, for example, Examples 19-22).

The term “treatment” as used herein indicates any activity that is partof a medical care for, or deals with, a condition, medically orsurgically.

The term “prevention” as used herein indicates any activity whichreduces the burden of mortality or morbidity from a condition in anindividual. This takes place at primary, secondary and tertiaryprevention levels, wherein: a) primary prevention avoids the developmentof a disease; b) secondary prevention activities are aimed at earlydisease treatment, thereby increasing opportunities for interventions toprevent progression of the disease and emergence of symptoms; and c)tertiary prevention reduces the negative impact of an alreadyestablished disease by restoring function and reducing disease-relatedcomplications.

The term “condition” as used herein indicates a physical status of thebody of an individual (as a whole or as one or more of its parts), thatdoes not conform to a standard physical status associated with a stateof complete physical, mental and social well-being for the individual.Conditions herein described include but are not limited disorders anddiseases wherein the term “disorder” indicates a condition of the livingindividual that is associated to a functional abnormality of the body orof any of its parts, and the term “disease” indicates a condition of theliving individual that impairs normal functioning of the body or of anyof its parts and is typically manifested by distinguishing signs andsymptoms.

The term “individual” as used herein in the context of treatmentincludes a single biological organism, including but not limited to,animals and in particular higher animals and in particular vertebratessuch as mammals and in particular human beings.

The wording “selective”, “specific” “specifically” or “specificity” asused herein with reference to the binding of a first molecule to secondmolecule refers to the recognition, contact and formation of a stablecomplex between the first molecule and the second molecule, togetherwith substantially less to no recognition, contact and formation of astable complex between each of the first molecule and the secondmolecule with other molecules that may be present. Exemplary specificbindings are antibody-antigen interaction, cellular receptor-ligandinteractions, polynucleotide hybridization, enzyme substrateinteractions etc. The term “specific” as used herein with reference to amolecular component of a complex, refers to the unique association ofthat component to the specific complex which the component is part of.The term “specific” as used herein with reference to a sequence of apolynucleotide refers to the unique association of the sequence with asingle polynucleotide which is the complementary sequence. By “stablecomplex” is meant a complex that is detectable and does not require anyarbitrary level of stability, although greater stability is generallypreferred. The term “selective” “specific” “specifically” or“specificity” as used herein with reference to a chemical or biologicalactivity of a first molecule to second molecule of a certain bacteria orgroup of bacteria refers to the ability of the first molecule to directthe activity towards the second molecule, together with substantiallyless to no activity between the first molecule and molecules that may bepresent of organisms other than the bacteria or group of bacteria.

In some embodiments, the method for treating and/or preventing abacterial infection in an individual comprises inactivation ofphenazines and/or one or more phenazine related pathways of the bacteriaas describe in any of the above embodiments. In particular, theinactivation of the phenazines and/or one or more phenazine relatedpathways of the bacteria performed as describe in any of the aboveembodiments will be recognized by the skilled person as not interferingin a deleterious manner with the normal biochemical pathways of theindividual.

In some embodiments, a method and system for identifying anantimicrobial is described. The method comprises contacting a microbewith a candidate agent and detecting the ability of the candidate agentof inactivating a phenazine and/or a phenazine related pathway in thebacteria. The system comprises one or more microbes and one or moreagents capable of detecting phenazine and/or phenazine related pathways.

An “antimicrobial” as described herein indicates a substance that killsor inhibits the growth of microorganisms such as bacteria, fungi, orprotozoans. Antimicrobial either kills microbes (microbiocidal) orprevent the growth of microbes (microbiostatic)

The terms “detect” or “detection” as used herein indicates thedetermination of the existence, presence or fact of a target in alimited portion of space, including but not limited to a sample, areaction mixture, a molecular complex and a substrate. The “detect” or“detection” as used herein can comprise determination of chemical and/orbiological properties of the target, including but not limited toability to interact, and in particular bind, other compounds, ability toactivate another compound and additional properties identifiable by askilled person upon reading of the present disclosure. The detection canbe quantitative or qualitative. A detection is “quantitative” when itrefers, relates to, or involves the measurement of quantity or amount ofthe target or signal (also referred as quantitation), which includes butis not limited to any analysis designed to determine the amounts orproportions of the target or signal. A detection is “qualitative” whenit refers, relates to, or involves identification of a quality or kindof the target or signal in terms of relative abundance to another targetor signal, which is not quantified.

In some embodiments, the method for identifying an antimicrobial furthercomprises contacting the microbe with an antibiotic and/or an additionalantimicrobial to the individual.

In some embodiments, the one or more agents of inactivating a phenazineand/or a phenazine related pathway is the agent according to firstaspect of the disclosure.

In some embodiments, the system for identifying an antimicrobialcomprises one or more microbes and one or more agents capable ofdetecting phenazine and/or phenazine related pathways for simultaneouscombined or sequential use in the method according to the first andsecond aspects of the disclosure.

In some embodiments, an antimicrobial is described. The antimicrobialcomprises one or more agents able to inactivate a phenazine and/or aphenazine related pathway in the bacteria to reduce antibioticresistance and/or survivability of bacteria and optionally a compatiblevehicle for effective administrating and/or delivering of the one ormore agents to an individual.

In some embodiments, a pharmaceutical composition for the treatment ofcystic fibrosis is described. The pharmaceutical composition for thetreatment of cystic fibrosis comprises one or more agents able toinactivate a phenazine and/or a phenazine related pathway in thebacteria to reduce survivability of bacteria. In some embodiments thepharmaceutical composition for the treatment of cystic fibrosiscomprises an Fe(II) chelator and an Fe(III) chelator.

In some embodiments the pharmaceutical composition for the treatment ofcystic fibrosis further comprises a suitable vehicle for effectiveadministrating and/or delivering of the one or more agents to anindividual.

In some embodiments, the Fe(II) chelator is Ferrozine® and is comprisedin the pharmaceutical composition in an amount ranging between about 10and about 1000 μM. In some embodiments, the Fe(III) chelator isconalbumin and is comprised in the pharmaceutical composition in anamount ranging between 10-1000 μM (see, for example, Examples 1-5 and19-22).

In some embodiments, the pharmaceutical composition comprises Ferrozine®in an amount ranging between about 10 and about 1000 μM and conalbuminFerrozine® in an amount ranging between about 10 and about 1000 μM (see,for example, Examples 1-5 and 19-22).

In some embodiments, the pharmaceutical composition comprises Ferrozine®in an amount of approximately 200 μM (see, for example, Examples 1-5 and19-22).

In some embodiments, the pharmaceutical composition comprises conalbuminin an amount of approximately 100 μM (see, for example, Examples 1-5 and19-22).

In some embodiments, the pharmaceutical composition comprising acombination of the Fe(II) chelator and the Fe(III) chelator reducesbiofilm accumulation by greater than approximately 20%. In someembodiments, the pharmaceutical composition comprising the combinationof the Fe(II) chelator and the Fe(III) chelator reduces biofilmaccumulation by approximately greater than 20% or by approximately 50%(see, for example, Examples 19-22).

The term “vehicle” as used herein indicates any of various media actingusually as solvents, carriers, binders or diluents for PSA comprised inthe composition as an active ingredient.

In some embodiments, the antimicrobial further comprises and antibioticand/or an additional antimicrobial.

In some embodiments, the vehicle is a pharmaceutically acceptablevehicle and the composition is a pharmaceutical composition.

In particular some embodiments, the one or more agents can be includedin pharmaceutical compositions together with an excipient or diluent andoptionally with one or more antibiotics and/or other antimicrobial.

The term “excipient” as used herein indicates an inactive substance usedas a carrier for the active ingredients of a medication. Suitableexcipients for the pharmaceutical compositions herein disclosed includeany substance that enhances the ability of the body of an individual toabsorb the one or more agents. Suitable excipients also include anysubstance that can be used to bulk up formulations with the one or moreagents to allow for convenient and accurate dosage. In addition to theiruse in the single-dosage quantity, excipients can be used in themanufacturing process to aid in the handling of the one or more agents.Depending on the route of administration, and form of medication,different excipients may be used. Exemplary excipients include but arenot limited to anti-adherents, binders, coatings disintegrants, fillers,flavors (such as sweeteners) and colors, glidants, lubricants,preservatives, sorbents.

The term “diluent” as used herein indicates a diluting agent which isissued to dilute or carry an active ingredient of a composition.Suitable diluent include any substance that can decrease the viscosityof a medicinal preparation.

As disclosed herein, the antimicrobial agents herein described can beprovided as a part of systems to perform any methods, including any ofthe assays described herein. The systems can be provided in the form ofarrays or kits of parts. An array, sometimes referred to as a“microarray”, can include any one, two or three dimensional arrangementof addressable regions bearing a particular molecule associated to thatregion. Usually, the characteristic feature size is micrometers.

In a kit of parts, the antimicrobial agent, candidate agents and otherreagents to perform the method can be comprised in the kitindependently. The antimicrobial agent can be included in one or morecompositions, and each capture agent can be in a composition togetherwith a suitable vehicle.

Additional components can include labeled molecules and in particular,labeled polynucleotides, labeled antibodies, labels, microfluidic chip,reference standards, and additional components identifiable by a skilledperson upon reading of the present disclosure. The terms “label” and“labeled molecule” as used herein as a component of a complex ormolecule referring to a molecule capable of detection, including but notlimited to radioactive isotopes, fluorophores, chemiluminescent dyes,chromophores, enzymes, enzymes substrates, enzyme cofactors, enzymeinhibitors, dyes, metal ions, nanoparticles, metal sols, ligands (suchas biotin, avidin, streptavidin or haptens) and the like. The term“fluorophore” refers to a substance or a portion thereof which iscapable of exhibiting fluorescence in a detectable image. As aconsequence, the wording “labeling signal” as used herein indicates thesignal emitted from the label that allows detection of the label,including but not limited to radioactivity, fluorescence,chemiluminescence, production of a compound in outcome of an enzymaticreaction and the like.

In some embodiments, detection of a phenazine, and phenazine relatedactivities can be carried either via fluorescent based readouts, inwhich the labeled antibody is labeled with fluorophore, which includes,but not exhaustively, small molecular dyes, protein chromophores,quantum dots, and gold nanoparticles. Additional techniques areidentifiable by a skilled person upon reading of the present disclosureand will not be further discussed in detail.

In particular, the components of the kit can be provided, with suitableinstructions and other necessary reagents, in order to perform themethods here described. The kit will normally contain the compositionsin separate containers. Instructions, for example written or audioinstructions, on paper or electronic support such as tapes or CD-ROMs,for carrying out the assay, will usually be included in the kit. The kitcan also contain, depending on the particular method used, otherpackaged reagents and materials (i.e. wash buffers and the like).

In some embodiments, the antimicrobial agents herein described can beincluded in pharmaceutical compositions together with an excipient ordiluent. In particular, in some embodiments, disclosed arepharmaceutical compositions which contain at least one multi-ligandcapture agent as herein described, in combination with one or morecompatible and pharmaceutically acceptable vehicles, and in particularwith pharmaceutically acceptable diluents or excipients. In thosepharmaceutical compositions the multi-ligand capture agent can beadministered as an active ingredient for treatment or prevention of acondition in an individual.

The term “excipient” as used herein indicates an inactive substance usedas a carrier for the active ingredients of a medication. Suitableexcipients for the pharmaceutical compositions herein described includeany substance that enhances the ability of the body of an individual toabsorb the multi-ligand capture agents or combinations thereof. Suitableexcipients also include any substance that can be used to bulk upformulations with the peptides or combinations thereof, to allow forconvenient and accurate dosage. In addition to their use in thesingle-dosage quantity, excipients can be used in the manufacturingprocess to aid in the handling of the peptides or combinations thereofconcerned. Depending on the route of administration, and form ofmedication, different excipients can be used. Exemplary excipientsinclude, but are not limited to, anti-adherents, binders, coatings,disintegrants, fillers, flavors (such as sweeteners) and colors,glidants, lubricants, preservatives, sorbents.

The term “diluent” as used herein indicates a diluting agent which isissued to dilute or carry an active ingredient of a composition.Suitable diluents include any substance that can decrease the viscosityof a medicinal preparation.

Further advantages and characteristics of the present disclosure willbecome more apparent hereinafter from the following detailed disclosureby way or illustration only with reference to an experimental section.

EXAMPLES

The methods and systems and related compounds and compositions hereindisclosed are further illustrated in the following examples, which areprovided by way of illustration and are not intended to be limiting.

In particular, the following examples illustrate exemplary phenazinerelated pathways and related methods and systems according to thepresent disclosure. A person skilled in the art will appreciate theapplicability and the necessary modifications to adapt the featuresdescribed in detail in the present section, to additional solutions,methods and systems according to embodiments of the present disclosure.

The following materials and methods were used in performing theexperiments illustrated in the examples herein described.

Chemicals.

Phenazine-1-carboxylate (PCA) was purified from aerobic stationarycultures of P. fluorescens strain 2-79 (NRRL B-15132) [1] grown inKing's A medium [2] at 30° C., as previously described [3]. Pyocyanin(PYO) was purified from aerobic stationary cultures of P. aeruginosastrain UCBPP-PA14 [4] grown in LB at 37° C., as previously described[3]. Pyoverdin, purified according to the method described inAlbrecht-Gary et al. [5], and pyochelin, characterized and synthesizedaccording to the published procedures [6, 7], were provided by ProfessorSchalk's group (Institut de Recherche de l'Ecole de Biotechnologie deStrasbourg, IREBS FRE3211 CNRS/Université Strasbourg, France).Fe(OH)₃(s), referred to the Fe(III) mineral ferrihydrite, wassynthesized according to the method described in Schwertmann and Cornell[8], as previously described [3]. Substantially iron-free conalbumin,1,10-phenanthroline, hydroxylamine hydrochloride, ammonium acetate,ferrous ammonium sulfate, and carrier DNA for yeast transformation werepurchased from Sigma-Aldrich. All enzymes used for DNA manipulation werepurchased from New England Biolabs.

Strains, Plasmids, Rimers and Growth Conditions.

In some of the examples, the strains, plasmids, and primers that areused are listed in Tables 1 and 2. For planktonic and biofilmexperiments with P. aeruginosa PA14 strains, 0.3 g/L Bacto Tryptic SoyBroth (“1% TSB”, where 1% is relative to the usual concentration of TSBmedium (30 g/L); Becton Dickinson) was used as medium. Where indicatedwith the PA14 siderophore null strain (ΔpvdAΔpchE), 1.0 μM Fe(OH)₃(s),10 μM PCA or PYO, or 1.0 μM Fe(OH)₃(s) together with 10 μM PCA or PYOwas added to 1% TSB. Also as indicated with the PA14 wild type andfeoB::MAR2xT7 mutant (from the non-redundant PA14 mutant library [9]),10 μM PCA or PYO alone, 40 μg/ml iron-free conalbumin alone or togetherwith 10 μM PCA or PYO was amended to 1% TSB. Flow-cell biofilmexperiments with the PA14 wild type and phenazine deletion (Δphz)strains were also performed in 1% TSB supplemented with 10 μM PCA,respectively. To confirm that in contrast to PA14 wild type, theΔpvdAΔpchE strain was unable to produce pyoverdin and pyochelin duringplanktonic growth, an iron deficient MOPS-based medium [100 mM MOPS atpH 7.2, 20 mM succinate, 93 mM NH₄Cl, 43 mM NaCl, 2.2 mM KH₂PO₄, 1 mMMgSO₄] [modified from ref. 10] was used. These planktonic growthexperiments were performed in acid-washed iron-free sterile glassculture tubes or polypropylene flasks at 37° C. with vigorous shaking at250 rpm to generate aerobic conditions. To confirm that the PA14feoB::MAR2xT7 mutant was disrupted in ferrous iron transport, cells wereincubated shaking anaerobically in Amberlite-treated 1% TSB mediumcontaining 100 mM KNO₃, 50 mM glutamate, 1% glycerol, and 100 μM ironsource (either (NH₄)₂Fe(II)(SO₄)₂ or Fe(III)Cl₃) at 37° C. for 22 hours.Culture densities were followed at 500 nm (OD₅₀₀) in a Thermo Spectronic20D+ or Shimadzu UV-2450 spectrophotometer.

P. aeruginosa PA14 strains and Escherichia coli strains used for mutantconstruction were cultured in Luria-Bertani (LB, Fisher Scientific)medium at 37° C. E. coli BW29427 and 1-2155 were supplemented with 0.3mM diaminopimelic acid. The yeast Saccharomyces cerevisiae uracilauxotrophic strain InvSc1 (Invitrogen) for gap repair cloning [11, 12]was grown with yeast extract-peptone-dextrose (YPD medium: 1% Bactoyeast extract, 2% Bacto peptone, and 2% dextrose) at 30° C., andselections were performed with synthetic defined agar (SDA) mediumlacking uracil (URA, Qbiogene 4813-065). For selection and maintenanceof plasmids pMQ30 and derivatives, as well as pAKN69, gentamicin wasused at 15 μg/ml for E. coli and 75-100 μg/ml for P. aeruginosa,respectively. Selection and maintenance of E. coli containing pUX-BF13was carried out on 100 μg/ml ampicillin.

TABLE 1 Reference or Strain or plasmid Properties source Strains P.aeruginosa PA14 Clinical isolate UCBPP-PA14, wild type  [4] strain PA14Δphz PA14 with deletions of operons phzA1-G1 [13] and phzA2-G2 PA14ΔpvdAΔpchE PA14 with deletions of pvdA and pchE This study PA14 PA14with deletions of pvdA, pchE, and This study ΔphzΔpvdAΔpchE operonsphzA1-G1 and phzA2-G2 PA14-YFP PA14 constitutively expressing YFP from aL. E. P. Dietrich, Tn7 insertion created by introducing MIT plasmidpAKN69 PA14 Δphz-YFP PA14 Δphz constitutively expressing YFP, L. E. P.Dietrich, analogous to PA14-YFP MIT PA14 ΔpvdAΔpchE- PA14 ΔpvdAΔpchEconstitutively This study YFP expressing YFP plasmid pAKN69 PA14 PA14ΔphzΔpvdAΔpchE constitutively This study ΔphzΔpvdAΔpchE-YFP expressingYFP plasmid pAKN69 PA14 feoB::MAR2xT7 PA14 mutant with an insertion ofthe  [9] MAR2xT7 transposon in the PA14_56680 ORF, which is the homologof the PAO1 ORF PA4358. E. coli UQ950 DH5α λ pir host for cloning D. P.Lies, Caltech BW29427 Donor strain for conjugation W. M. Metcalf, UIUCβ-2155 Donor strain for conjugation [14] S. cerevisiae InvSC1 Ura⁻ forgap repair cloning Invitrogen Plasmids pMQ30 Yeast-based allelicexchange vector, sacB, [15] CEN/ARSH, URA3⁺, Gm^(R) pUX-BF13 R6Kreplicon-based helper plasmid, [16] providing the Tn7 transpositionfunctions in trans, which can only replicate when the pir gene issupplied in trans, Amp^(R) pAKN69 Transposon delivery plasmid containingthe [17] mini-Tn7(Gm)P_(A1/04/03)::eyfp fusion pYW01 pvdA deletionfragments cloned into This study pMQ30 pYW02 pchE deletion fragmentscloned into This study pMQ30

In some of the examples, P. aeruginosa PA14 and its derivatives wereused in this study. Strain DKN330, a ΔphzA1-G1 ΔphzA2-G2 deletion mutant[13] is unable to produce any phenazine and is referred to here asΔphz1/2. Plasmid pAKN69, containing the mini-Tn7(Gm)P_(A1/04/03)::eypfusion [18], was used to introduce a chromosomally-encoded constitutiveeYFP into PA14 wild type and Δphz1/2, resulting in strains DKN372 andDKN373, respectively.

In some of the examples, P. aeruginosa strain PA14 [4] was used, whichproduces approximately ten times more pyocyanin in LB batch culturesthan strain PAO1 [13]. The P. aeruginosa PA14 mutant containing theMAR2xT7 transposon inserted in the IdhA gene in a ΔexoU background wasobtained from a publicly available mutant library [19] and is mutant ID#5174. Generation of the P. aeruginosa PA14 ΔphzA1-1G1 ΔphzA2-2G2deletion mutant (hereafter referred to as the Δphz mutant) was describedpreviously [13]. P. aeruginosa PA14 wild type and mutants were grownaerobically at 37° C. in LB Broth, Miller (Fisher Scientific) ormodified MOPS synthetic medium [10]. Our modified MOPS synthetic mediumcontained 50 mM morpholinepropanesulfonic acid (MOPS, Sigma) at pH 7.2,93 mM NH₄Cl, 43 mM NaCl, 2.2 mM KH₂PO₄, 1 mM MgSO₄.7H₂O, and 3.6 μMFeSO₄.7H₂O. Unless otherwise noted, 50 mM D-glucose was added to thismedium as the sole carbon and energy source. Aerobic conditions weregenerated either through incubation with vigorous shaking at 250 rpm, orin a BioFlo 110 fermentor (New Brunswick Scientific) set to agitate at250 rpm and bubble with 100% air at a rate of 2 Uminute. Aerobic culturevolumes relative to vessel size are described below for specificexperiments. Culture densities were followed at 500 or 600 nm in aThermo Spectronic 20D+ or Beckman Coulter DU 800 spectrophotometer.Cultures with optical densities greater than 0.8 were diluted 1:10 infresh medium to allow accurate measurements. The method used to teststrains for the ability to survive via pyruvate fermentation isdescribed in supplementary material.

Strain Construction.

P. aeruginosa synthesizes two known siderophores, the strongerFe(III)-binding pyoverdin and the weaker Fe(III)-binding pyochelin [20].Applicant constructed the siderophore null strain by generating unmarkeddeletions of pyoverdin and pyochelin biosynthetic genes pvdA [21] andpchE [22] in PA14 wild type, respectively. Analogously, Applicantconstructed the phenazine-siderophore null strain in the PA14 phenazinenull strain (Δphz). Applicant first deleted pvdA and then pchE. HereApplicant describes the protocol for in-frame deletion of pvdA usingyeast gap repair cloning based on previously developed methods [11, 12,15, 23]. The 5′ and the 3′ regions (both ˜1 kb in lengths) of thesequence flanking pvdA were amplified using primer pairs pvdAKO1 (SEQ IDNO: 1)/pvdAKO2 (SEQ ID NO: 2) and pvdAKO3 (SEQ ID NO: 3)/pvdAKO4 (SEQ IDNO: 4), respectively (Table 2). These 5′ and 3′ flanking DNA fragmentsand the gapped plasmid vector pMQ30 were simultaneously introduced intothe yeast S. cerevisiae uracil auxotrophic strain InvSc1 (Invitrogen)for in vivo recombination [11, 12]. The plasmid pMQ30 is an allelicexchange vector for Gram-negative bacteria unable to support replicationof the ColE1 origin and contains CEN6/ARSH4 DNA sequences to supportreplication in S. cerevisiae, a URA3 yeast selectable marker, amulticloning site in a lacZα-allele for blue-white screening, an oriTfor conjugation-mediated plasmid transfer, a gentamicin-resistance gene(aacC1); and the counter-selectable marker sacB [15]. Recombinant yeastcells were selected for on medium deficient in uracil. Plasmids wereliberated from recombinant yeast and electroporated into E. coli UQ950,which was used as a host strain for plasmid replication [24].Transformants containing recombination products of pMQ30 with the PCRproducts were isolated by blue/white screening and gentamicin resistance[24], yielding the construct for deleting the PA14 pvdA gene. Thisplasmid, which is called pYW01 (Table 1) was purified from E. coliUQ950, transformed by heat shock into E. coli BW29497 (a diaminopimelicacid auxotroph), and then mobilized into PA14 (wild type or the Δphzmutant) via biparental conjugation [25]. PA14 (wild type and the Δphzmutant) single recombinants (merodiploid containing the intact and thedeleted pvdA gene) were isolated by selecting for gentamicin resistance.Resolution of the merodiploid was performed by selection on 10% sucrose,followed by PCR-based screening for loss of the wild type gene toisolate the pvdA deletion mutants (referred to as ΔpvdA and ΔphzΔpvdA).The deletion of pchE from strains ΔpvdA and ΔphzΔpvdA were generated thesame way, using primer pairs pchEKO1 (SEQ ID NO: 5)/pchEKO2 (SEQ ID NO:6) and pchEKO3 (SEQ ID NO: 7)/pchEKO4 (SEQ ID NO: 8) (Table 2). Besidesusing PCR-based diagnosis, the mutants ΔpvdAΔpchE and ΔphzΔpvdAΔpchEwere further confirmed by their inability to produce pyoverdin orpyochelin when growing the mutant in iron deficient MOPS-based medium,in contrast to the wild type and the Δphz strains, respectively (see‘Analyses of Phenazines and Siderophores’ for pyoverdin and pyochelindetections).

TABLE 2 Primer Sequence (5′ to 3′) pvdAKO1CCA GGC AAA TTC TGT TTT ATC AGA CCG CTTCTG CGT TCT GAT AGC GCT GGA ACT CGC CAC (SEQ ID NO: 1) pvdAKO2GCT TCA GGT GCT GGT ACA GTG CCT GAG TCA TTT CCA GTT CC (SEQ ID NO: 2)pvdAKO3 GGA ACT GGA AAT GAC TCA GGC ACT GTA CCAGCA CCT GAA GC (SEQ ID NO: 3) pvdAKO4GGA ATT GTG AGC GGA TAA CAA TTT CAC ACAGGA AAC AGC TCT GAA GCC GAT GTT GAC CAC (SEQ ID NO: 4) pvdEKO1CCA GGC AAA TTC TGT TTT ATC AGA CCG CTTCTG CGT TCT GAT CTG ATC CTC GTG CAG AGC (SEQ ID NO: 5) pvdEKO2GGT CTG CAC CTG CAA GTG CAG GGC GGT ACG GGA ATC (SEQ ID NO: 6) pvdEKO3GAT TCC CGT ACC GCC CTG CAC TTG CAG GTG CAG ACC (SEQ ID NO: 7) pvdEKO4GGA ATT GTG AGC GGA TAA CAA TTT CAC ACAGGA AAC AGC TCG TCA GGT TGA GAC AGA ACG (SEQ ID NO: 8)

To follow P. aeruginosa biofilm development using confocal microscopy,YFP-labeled PA14 strains were generated by introducing achromosomally-encoded constitutive EYFP into the wild type, thephenazine null strain (Δphz), the siderophore null strain (ΔpvdAΔpchE),and the phenazine-siderophore null strain (ΔphzΔpvdAΔpchE),respectively. Plasmid pAKN69 containing themini-Tn7(Gm)P_(A1/04/03)-eyfp fusion was used for this purpose [17].This plasmid was cloned into E. coli BW29427, and then mobilized intoeach PA14 strain via triparental mating with the helper plasmid pUX-BF13(carrying the transposase genes) in E. coli β-2155, as describedpreviously [16, 26]. PA14 transformants with YFP constructs wereselected with gentamicin and confirmed by YFP fluorescence.

Biofilm Experiments.

A flow cell system was constructed for biofilm experiments. The size ofeach flow channel was 1.5×4×34 mm; continuous flow of 1% TSB-basedbiofilm medium (with or without the respective additives detailed in theResults section) at the rate of 3 ml/h was supplied with a Watson-Marlowperistaltic pump; the temperature for biofilm growth was 22° C. An earlystationary phase culture grown in 10% TSB was diluted to an OD₅₀₀ of 0.1in biofilm control medium (1% TSB). Each flow cell was then inoculatedwith 300 μl of the diluted culture by injection with a 1 ml syringe. Inorder to allow cells to attach to the glass surface, the flow wasarrested for 1.5 hours and then resumed throughout the length of eachexperiment (up to 6 days).

To image biofilms, confocal laser scanning microscopy (CLSM) with aLeica TCS SPE inverted microscope was used. For PA14 strainsconstitutively expressing EYFP (wild type, Δphz, ΔpvdADpchE, andΔphzΔpvdAΔpchE), 3-dimensional fluorescence images were acquired usingan excitation wavelength of 488 nm with constant intensity andcollecting emission in the range of 510-618 nm. For the PA14feoB::MAR2xT7 mutant, images were obtained using differentialinterference contrast (DIC) mode. To assure images used for comparisonsof biofilm formation were representative and reproducible, multiplefields of view were acquired over time with a 10× dry objective in eachflow cell within a single experimental set, and at least 4 independentexperimental sets were performed. Fluorescence and DIC images wereprocessed using Bitplane Imaris and NIH imageJ software. In most cases,fluorescence-based multiple biofilm image stacks (spaced 1-2 μm apart)were analyzed using the autoCOMSTAT software, a modified version of theCOMSTAT biofilm evaluation package by Heydorn et al. [27, 28]. For eachimage a global threshold was calculated using the Robust AutomatedThreshold Selection algorithm with a critical-size setting of 20 μm, andconnected-volume filtering was performed with a connectivity setting of18 to remove free-floating biomass. Substratum coverage calculationswere based on the first 3 μm above the substratum. The area of eachanalyzed image was 3.03×10⁵ μm² and results from measurements of 1-6images for each strain and treatment were averaged and sample standarddeviations were calculated. The biofilm parameters reported here arebiovolume per image area (referred to as biomass), substratum coverage,maximum height, and average height of the biomass, which excludes anyarea not covered by cells.

Iron Analysis.

In some examples, the total iron concentrations in TSB-based media andthe mineral ferrihydrite [Fe(OH)₃(s)] suspensions were analyzed by thephenanthroline assay according to published protocols [29]. In summary,complete reduction of Fe(III) (soluble and/or mineral forms) to solubleFe(II) was achieved by adding the reductant hydroxylamine hydrochlorideto acidified samples. 1,10-phenanthroline and the pH buffer ammoniumacetate were then added and allowed enough time to fully develop anorange-red Fe(II)-phenanthroline complex at pH 3.5. The total ironconcentrations reflected by the colored Fe(II) complex were calculatedbased on the absorbance readings at 510 nm in a Shimadzu UV-2450spectrophotometer. Iron in sterile, aerobic TSB-based media should bepresent as the oxidation state +3 even though the specific Fe(III) formsare unknown.

In some examples, ferrous iron and total iron were quantified using theFerrozine® assay [56]. Briefly, 50 μL of sputum filtrate was added to 50μL of 1M HCl to quantify Fe(II). For total iron, 50 μL was treated with50 μL of 10% hydroxylamine hydrochloride in 1M HCl to reduce Fe(III) toFe(II). Samples were added to 100 μL of Ferrozine® (0.1% w/v in 50%ammonium acetate), incubated for 15 min, and absorbance was measuredspectrophotometrically at 562 nm. Ferrous ammonium sulfate was used asthe iron standard. Ferrozine® was also used to determine the Fe(II)composition of the trypticase soy broth (TSB) growth medium.

In some examples, samples were also analyzed by inductively coupledplasma mass spectrometry (ICP-MS), a highly sensitive mass spectrometrymethod capable of metal determination below one part per trillion.Briefly, 50 μL of filtrate was digested in 100 μL 8N nitric acid, andbrought to a total of 1.5 mL in 5% nitric acid/indium standard. Sampleswere analyzed on an Agilent 7500 cx equipped with a reaction cell, usingHe (2 mL/min) and H2 (2.5 mL/min) as reaction gases. Fe concentrationswere calculated using ⁵⁶Fe and ⁵⁷Fe signal intensities.

ICP-MS Versus Ferrozine® Determination of Total Iron.

In some examples, the accuracy and precision of the Ferrozine® assay arecompromised as the proportion of Fe(II) increases, leading tooverestimations of total iron concentrations [58]. To control for this,total sputum iron was quantified using ICP-MS. As expected, comparisonof the two methods revealed a higher estimate of total iron using thecolorimetric approach (FIG. 18). On average, Ferrozine® measurementswere 30% greater than those obtained using ICP-MS, indicating thatFe(II) levels determined here were higher than those present in sputum.For this reason, a 30% reduction was applied to all reported ferrousiron sputum concentrations in FIG. 17 Panel A. Despite this conservativereduction, sputum Fe(II) levels are frequently greater than those usedin our chelation experiments (˜10 μM), which were sufficient to impedeFe(III)-chelation treatment.

Analyses of Phenazines and Siderophores.

Filtrates (0.2 m pore size) were prepared from biofilm effluents orplanktonic cultures. For characterizing and quantifying phenazines andthe siderophore pyochelin, filtrates were directly loaded onto a BeckmanSystem Gold reverse-phase HPLC with a diode array UV-VIS detector and aWaters Symmetry® C18 Analytical column (5 μm particle size; 4.6×250 mm).Analysis was performed in a gradient of water-0.1% trifluoroacetic acid(TFA, solvent A) to acetonitrile-0.1% TFA (solvent B) at a flow rate of1.0 ml/min using the following method: for 0 to 1 min chromatography wasin a linear gradient from 100% solvent A to 15% solvent B, for 1 to 12min in a linear gradient to 58% solvent B, for 12 to 13 min in a lineargradient to 70% solvent B, for 13 to 25 min in a linear gradient to 85%solvent B, for 25 to 26 min in a linear gradient to 100% solvent A, for26 to 29 min in 100% solvent A. Phenazines (e.g., PCA, PYO,phenazine-1-carboxamide, and 1-hydroxyphenazine) and pyochelin that areknown to be potentially produced by P. aeruginosa can all be detectedbased on their characteristic absorption wavelengths and retention timesas long as their concentrations are higher than 0.05-0.1 μM [3, 13, 30,31].

The same HPLC instrument was used for analyzing the siderophorepyoverdin, with gradient profiling and sample preparation described indetail by Bultreys et al. [32]. In summary, filtrates withFe(III)-chelated pyoverdin(s) at pH 5.0 were prepared for HPLC analysisby adding FeCl₃ into samples followed by filtration (0.2 μm pore size)and pH adjustment. Analysis was carried out in a gradient profiling withsolvent A as water-17 mM NaOH-acetic acid at pH 5.0, and solvent B asacetonitrile (solvent B) [32]. For samples with pyoverdin being releasedat concentrations higher than 0.1 μM, a single Fe(III)-pyoverdin peakwas detected at 403 nm. In addition, iron-free pyoverdin in filtered(0.2 μm pore size) biofilm effluents was analyzed using a fastfluorescence-based method by a BioTek Synergy 4 fluorescence platereader with a Xenon Flash light source at the specificexcitation/emission wavelength set of 405 nm/455 nm [33]. Controlexperiments and independent HPLC analysis confirmed that the measuredfluorescence signal was predominantly contributed by pyoverdin and hencecan be used for its detection and quantification.

Cell-Surface Attachment.

Attachment was analyzed using phase contrast imaging on a Leica confocalmicroscope. Stationary-phase cultures were diluted 1:50 in 10% LB and0.5 ml of this suspension were pipetted into a sterile chambered system(Lab-Tek, Rochester N.Y.) with a borosilicate cover glass bottom. After0.5 h or 4 h incubation at 22° C., unattached cells were discarded bygently replacing the supernatant with fresh medium, and attached cellswere counted. Six fields of view for each strain and condition wereanalyzed and the percent of the surface covered by attached cells wasestimated using Adobe Photoshop.

Motility Assays.

Swimming, swarming and twitching motilities were determined aspreviously described [34].

Flow cell biofilms were grown under constant flow at 22° C. in 1.5×4×34mm flow cells. Continuous flow of 10% LB was supplied with a peristalticpump at a constant rate of 3 ml h⁻¹. An early stationary-phase culturewas diluted to an optical density at 500 nm of around 0.1 and 300 μlwere inoculated into the flow cell. Strains expressing eyfpconstitutively were used to visualize the biofilms. Upon inoculation,cells were allowed to attach in the absence of flow for 1.5 h beforeflow was resumed. Developing biofilms were imaged in 3 dimensions usinga Leica confocal microscope. eYFP was excited with a 488 nm laser beamkept at constant intensity throughout the experiment, and emission from510 to 618 nm was collected. Routinely, the Applicant observed that thedistribution of bacterial cells throughout the colonized surface varieddepending on the region of the flow cell, probably due to factors suchas flow or accumulation of planktonic cells. To assure reproducibility,all images were acquired from an area set in the middle of the flowcell. Three images of duplicate flow cell lines were recorded and atleast two independent experiments were performed.

Colony biofilms were grown on agar containing 1% tryptone as previouslydescribed [35]. Three colonies from independent spottings weredocumented for 8 days using an Epson scanner.

Preparation of Pyocyanin for Reduction Assays and NADH/NAD⁺ Studies.

To maximize pyocyanin yields from P. aeruginosa cultures, a mutant,strain DKN370 was utilized, which contains two copies of the gene phzM.PhzM converts phenazine-1-carboxylic acid to the precursor forpyocyanin, 5-methylphenazinium carboxylate [36]. Purification ofpyocyanin by organic extractions was carried out as described previously[13]. HPLC analysis verified the purity of pyocyanin after theextraction step, so the HPLC purification step described in [13] wasomitted. Purified pyocyanin was dissolved in MOPS buffer (MOPS syntheticmedium without FeSO₄, MgSO₄, or glucose), and filtered (0.2 μm).

Whole Cell Suspension Assay for Pyocyanin Reduction.

Cell culture samples were concentrated or diluted in filtrates ofsupernatants from the same culture to normalize optical density at 600nm to 0.8. In an anaerobic chamber, the samples were transferred tocuvettes, and an anoxic solution of oxidized pyocyanin (in MOPS buffer)was added for a final pyocyanin concentration of about 0.1 mM. Thecuvettes were stoppered to minimize oxygen exposure. Pyocyanin reductionwas then followed as a decrease in absorbance at 690 nm over time in aDU 800 Beckman Coulter spectrophotometer. The rate of reduction could becalculated by converting the change in absorbance to μmol pyocyaninusing the extinction coefficient for pyocyanin at this wavelength(ε=4310 M⁻¹ cm⁻¹ at pH 7 [37]) and the volume of sample in the cuvette(1 ml).

Quantification of Pyocyanin.

Pyocyanin concentrations in filtrates (0.2 μm pore) from LB and MOPSsynthetic medium cultures were quantified as described previously [13].Briefly, absorbance in LB culture filtrates was measuredspectrophotometrically at 690 nm and pyocyanin concentrations werecalculated using the extinction coefficient for pyocyanin (above).Pyocyanin concentrations in 200 μl sample filtrates from MOPS syntheticmedium cultures were determined by HPLC analysis on a Waters SymmetryC18 reverse-phase column with a gradient method (water vs. acetonitrilecontaining 0.1% trifluoroacetic acid) and calculated based on absorbancevalues for purified standards diluted into MOPS buffer.

Extraction and Quantification of Intracellular NADH and NAD⁺.

Extraction of NADH and NAD⁺ was carried out according to the methoddescribed in San et al. [38]. Two×1 ml of culture were sampled into twoseparate microcentrifuge tubes and centrifuged at 16,000×g for 1 min.Supernatant was removed and pellets were resuspended in 300 μl of 0.2 MNaOH (for NADH extraction) or 0.2 M HCl (for NAD⁺ extraction). Theseextracts were incubated for 10 min at 50° C., then for 10 min on ice.While vortexing, 300 μl of 0.1 M HCl (for NADH) or 0.1 M NaOH (for NAD⁺)were added drop wise to neutralize the solutions. They were thencentrifuged for 5 min at 16,000× g. Supernatants were transferred tofresh tubes and stored at −80° C. until quantification.

Relative or absolute NADH and NAD⁺ were quantified using a modification[38] of the enzyme cycling assay developed by Bernofsky and Swan [39],adapted for measurement in a microtiter plate. A master reagent mix wasprepared with 1× Bicine buffer (2.0 M, pH 8.0), 8× water, 1×80 mM EDTA,2×100% EtOH, 2×4.2 mM thiazolyl blue (MTT), and 4×16.6 mM phenazineethosulfate. The reagent mix was warmed to 30° C., then 90 μl aliquotswere dispensed into individual wells of a 96-well microtiter plate. Fiveμl of standard or sample were added to each well, then the cyclingreaction was started by the addition of 5 μl of alcohol dehydrogenase(Sigma # A-3263) prepared at 347 units/ml in 0.1 M Bicine (pH 8.0). Themicrotiter plate was incubated at 30° C., mixed by brief shaking, andread every 30-60 seconds for absorbance at 570 nm, which is the spectralpeak of MTT that increases upon reduction. Slopes arising from plots ofabsorbance at 570 nm over time were generated for NADH and NAD⁺standards as well as all samples. Standard curves were used to calculatethe absolute concentrations in μM, and values were normalized to opticaldensity of the original cell culture sample.

Relative Quantification of Dissolved Oxygen in Batch Cultures.

Oxygen was measured in batch fermentor cultures using a Clark electrode[40]. The electrode was calibrated such that the reading obtained by thecomputer without the probe attached was equal to zero, while the initialreading for the uninoculated medium (after aeration and agitation for 12hours) was set to 100 percent.

Analysis of Small Organic Acids in Culture Filtrates.

Two hundred μl were sampled from MOPS-glucose cultures (10 ml in an18×150 mm test tube) at regular intervals and filtered (0.2 μm pore). Incases where repeated sampling from the same culture would alter thetotal culture volume by more than 10%, multiple identical cultures wereinoculated from the same pre-culture and sampled sequentially. Twenty μlof each filtrate were loaded onto a Bio-Rad Aminex HPX-87H column(300×7.8 mm) and subjected to an isocratic method in 5 mM H₂SO₄ at 35°C., using a Waters HPLC system. Compounds were detected by UV absorbanceat 210 nm. Absolute concentrations of pyruvate were calculated using astandard curve for pyruvate diluted in MOPS buffer. The identity of thepyruvate peak was verified by co-elution of an internal standard.

Sputum Collection.

In some experiments, sputum was obtained by expectoration and wasimmediately flash frozen in liquid nitrogen to minimize oxidation.

Sputum Processing.

In some examples, frozen sputum samples were allowed to thaw in ananaerobic chamber. Sputum was disrupted using a syringe and was furtherhomogenized by vortexing in an equal volume of anaerobic 50 mM HEPESbuffer. Sputum was centrifuged at 8,000×g for 10 min and supernatantswere filtered through 0.22 μM columns for 20 minutes at 10,000× g.Filtrates were analyzed (anaerobically) for iron content. Whensufficient material was obtained, 200 μL of filtrate was stored at −80°C. for ICP-MS analysis.

MBEC Assay for Biofilm Prevention and Dissolution.

In some examples, biofilm prevention and dissolution were measured via ahigh-throughput biofilm assay (MBEC Physiology and Genetics Assay)consisting of a 96-well plate into which a 96-peg plastic lid fits. Thislid also fits over a standard 96-well plate, which was subsequently usedto test the efficacy of iron chelators. Inoculum was prepared bydiluting (30-fold) a 1.0 McFarland suspension of P. aeruginosa PA14 inTSB. 150 μL was dispensed into each of the 60 inner wells, while 200 μLof sterile trypticase soy broth (TSB) was placed in each perimeter well.For dissolution experiments, plates were incubated at 37° C. for 24 h,and were transferred to an anaerobic chamber for 24 h at 37° C. inanaerobic TSB containing 50 mM KNO₃. Biofilms were then exposed toconalbumin and/or Ferrozine® for 24 h. Each treatment was complementedwith the addition of 80 μM ferrous ammonium sulfate. After treatment,lids were rinsed once in 50 mM HEPES, air dried for 10 min andquantified by crystal violet staining [57]. For biofilm prevention,anaerobic inoculum was amended with conalbumin and/or Ferrozine®. Mediawas replaced every 24 h by transferring the MBEC lid to a sterile platecontaining growth media+/− treatments, and biofilms were allowed todevelop for 168 h. Biomass was quantified as described above. Biologicaltriplicates and six technical replicates (n=18) were used for eachtreatment.

Statistical Analysis.

In some examples, two-tailed student t-tests were used for pairwisecomparisons between patients groups (FIG. 17 Panel A) and chelatortreatments relative to untreated controls (FIG. 17 Panel A). Pairwisecomparisons were also performed between chelator treatments and thosecomplemented with 80 μM Fe(II). In all cases, P<0.05 was consideredstatistically significant.

Example 1: Phemazine-1-Carboxylic Acid (PCA) Cam Work Together with theSiderophore Pyoverdin in Promoting Biofilm Development

Biofilm development in P. aeruginosa PA14 wild type, a phenazine nullstrain (Δphz), a siderophore null strain (ΔpvdAΔpchE), and aphenazine-siderophore null strain (ΔphzΔpvdAΔpchE) under a flow of 1%TSB medium was monitored over 4 days. As illustrated in FIG. 1, wildtype biofilm formation proceeded in the typical stages such thatbacteria initially attached to the abiotic glass surface, clustered intomicrocolonies by day 1, which enlarged over time and eventually maturedby day 4; the Δphz mutant, despite initially attaching equally well,formed fewer and smaller microcolonies over time in comparison to thewild type; the ΔpvdAΔpchE and ΔphzΔpvdAΔpchE mutants showed equallysevere biofilm defects, and failed to develop microcolonies even 4 daysafter initial attachment (FIG. 1A). More quantitatively, the wild typebiomass was only marginally higher than those of three other strains atday 1, but by day 4 was about 3-4 times greater than that of Δphz and40-60 times those of ΔpvdAΔpchE and ΔphzΔpvdAΔpchE (FIG. 1B, Table 3).By day 4, the difference in total biomass between the wild type and Δphzmutant biofilms reflects variation mainly in thickness. On the otherhand, the biofilm thickness observed for ΔpvdAΔpchE and ΔphzΔpvdAΔpchEwas about the same as that of Δphz; their extremely low total biomassresults primarily from a lack of surface coverage (Table 3).

TABLE 3 Avg. thickness Max. biofilm Total biomass Substratum of biomassthickness Time PA14 strain n (μm³/μm²) coverage (%) (μm)¹ (μm) Day 1 WT6 0.83 ± 0.11  7.1 ± 0.8 13.2 ± 0.4 40 Δphz 6 0.18 ± 0.03  1.9 ± 0.311.3 ± 0.8 35 ΔpνdAΔpchE 6 0.38 ± 0.08  4.5 ± 0.9 10.8 ± 0.4 28ΔphzΔpνdAΔpchE 6 0.59 ± 0.08  5.7 ± 0.9 13.0 ± 0.6 37 Day 2 WT 5 2.54 ±0.41 14.0 ± 1.0 19.9 ± 3.4 89 Δphz 6 1.04 ± 0.15 10.0 ± 0.9 13.0 ± 1.960 ΔpνdAΔpchE 6 0.50 ± 0.08  4.5 ± 0.7 12.2 ± 0.2 33 ΔphzΔpνdAΔpchE 60.79 ± 0.13  6.3 ± 0.8 13.4 ± 0.5 31 Day 4 WT 3 8.10 ± 0.75 28.1 ± 0.628.9 ± 3.0 95 Δphz 4 2.37 ± 0.26 23.1 ± 3.3 13.8 ± 0.7 44 ΔpνdAΔpchE 20.20 ± 0.09  1.5 ± 0.1 13.8 ± 4.3 47 ΔphzΔpνdAΔpchE 4 0.13 ± 0.05  1.0 ±0.3 14.0 ± 0.9 36 All values are means of results of n images ± standarderror of the mean. Each image covers an area of 3.03 × 10⁵ μm².¹Calculated for the area coverd by biomass.

To further correlate phenazine and/or siderophore production withbiofilm development, simultaneous analysis of the compounds beingreleased into the biofilm effluents for the wild type, Δphz andΔpvdAΔpchE strains was performed (FIG. 1C). The wild type started torelease measurable levels of PCA (the precursor phenazine produced byall phenazine-making pseudomonads) and the siderophore pyoverdinbeginning on day 2. Concomitant with biofilm development, both PCA andpyoverdin concentrations increased with time, with a dramatic rise inthe PCA concentration by day 4.

Surprisingly, no other phenazines, including the blue colored pyocyanin(PYO), ever reached concentration levels above their respective HPLCdetection limits (0.05-0.1 μM) under our biofilm growth conditions.Given that PYO is a highly diffusible zwitterion at circumneutral pH,Applicant interpreted the absence of PYO in the biofilm effluents toreflect little or no production rather than its retention in the biofilmmatrix. The Δphz mutant started to release a measurable level ofpyoverdin on day 2 and the pyoverdin concentration increased along withbiofilm development. By day 4, despite the fact that the Δphz mutantreleased more pyoverdin than the wild type, it generated 3-4 times lessbiofilm biomass. Adding 10 μM PCA (comparable to the levels detected inwild type day 4 biofilm effluents) to the medium flow of the Δphz mutantthroughout the experimental timescale increased the biofilm biomass fourtimes by day 6, resulting in a biomass similar to the wild type. TheΔpvdAΔpchE mutant that failed to form biofilms did not releasephenazines until day 4, and even then only at trace levels, consistentwith one of the two phenazine biosynthetic operons being quorum-sensingregulated and that phenazines are typically only released at a high celldensity.

The lack of phenazine production by ΔpvdAΔpchE matches our observationthat ΔpvdAΔpchE and ΔphzΔpvdAΔpchE show equally severe biofilm defects.Collectively, these data indicate that both siderophores and phenazines(particularly PCA under these conditions) promote biofilm formation.

In view of the above it is expected that inactivation of genes involvedin the production of phenazines result in inactivation of phenazinemediated biofilm formation in P. aeruginosa or other bacteria.

Example 2: PCA can Promote Biofilm Formation by Generating Fe(II) is theAbsence of Siderophores

It is well established that pyoverdin promotes P. aeruginosa biofilmformation by facilitating iron acquisition. Effective iron acquisitionis essential for biofilm development because more iron is required thanfor planktonic growth because it acts as a signal to trigger thetransition from a motile to a sessile state. From the phenanthrolineassay, the total iron concentration is calculated to be 0.2 μM in 1%TSB.

Because the medium was prepared aerobically at pH 7, the oxidation stateof iron originally present in the medium should be +3, even though thespecific Fe(III) forms are unknown. It is commonly believed thatbioavailable iron in the micromolar concentration range is required foroptimal bacterial growth, and hence the iron content in 1% TSB verges onthe lower threshold. If the observed PCA-promoted biofilm formation(FIG. 1) were due to a stimulation in iron acquisition as a result ofphenazine-facilitated Fe(III) reduction to Fe(II), one would expect thatPCA would be able to circumvent the pyoverdin pathway if enough Fe(III)were provided.

To test whether PCA could rescue the biofilm defect in the PA14ΔpvdAΔpchE mutant in the presence of sparingly soluble Fe(III), theeffect of adding 1.0 μM ferrihydrite mineral suspension [Fe(OH)₃(s);K_(sp)=10^(−38.8) M [41]], 10 μM PCA, or 1.0 μM Fe(OH)₃(s) together with10 μM PCA to the base medium (1% TSB) was examined. Adding exogenous PCAwas necessary because, unlike the wild type, ΔpvdAΔpchE produced verylittle PCA on its own (FIG. 1C). Adding Fe(OH)₃(s) or PCA alone couldnot rescue biofilm formation by the ΔpvdAΔpchE mutant strain withstatistical significance (FIG. 2, Table 4). In contrast, when Fe(OH)₃(s)and PCA were added together, dramatic rescue was observed (FIG. 2, Table4).

This is consistent with prior abiotic experiments showing that somephenazines, including reduced PCA, can promote Fe(OH)₃(s) reduction overa broad pH range and liberate bioavailable Fe(II). The fact that addingPCA to 1% TSB can promote biofilm formation by the Δphz mutant (whichmakes pyoverdin but not phenazines) but not the ΔpvdAΔpchE mutant (whichcannot make pyoverdin, and is severely delayed in making phenazines;FIG. 1C) further supports the conclusion that PCA and pyoverdin bothcontribute to biofilm formation. However, even in the absence ofpyoverdin, PCA-promoted rescue can be achieved by adding just 1 μMFe(III) to the base medium originally with 0.2 μM total Fe (FIG. 2 Table4).

TABLE 4 Additive(s) to Avg. thickness Max. biofilm biofilm control Totalbiomass Substratum of biomass thickness Time medium (1% TSB) n (μm³/μm²)coverage (%) (μm)¹ (μm) Day 2 none ND 1.0 μm Fe(OH)₃(s) 4 0.03 ± 0.00 0.4 ± 0.1 10.7 ± 2.0 30  10 μm PCA 4 0.05 ± 0.01  0.6 ± 0.2 12.6 ± 1.432 1.0 μm Fe(OH)₃(s), 4 0.45 ± 0.21  6.3 ± 3.3 11.1 ± 1.2 30  10 μm PCA 10 μm PYO 3 0.09 ± 0.03  0.8 ± 0.3 13.5 ± 0.4 34 1.0 μm Fe(OH)₃(s), 40.57 ± 0.29  7.3 ± 3.7 10.3 ± 0.5 34  10 μm PYO Day 4 none ND 1.0 μmFe(OH)₃(s) 3 0.05 ± 0.01  0.5 ± 0.2 12.1 ± 2.3 30  10 μm PCA 4 0.19 ±0.09  2.9 ± 1.4 10.8 ± 0.9 34 1.0 μm Fe(OH)₃(s), 4 1.90 ± 1.08 12.5 ±6.4 15.8 ± 1.8 44  10 μm PCA  10 μm PYO 4 0.83 ± 0.22  9.9 ± 2.4 10.1 ±0.4 42 1.0 μm Fe(OH)₃(s), 4 3.16 ± 1.76 19.2 ± 9.2 10.8 ± 2.4 48  10 μmPYO Day 6 none 4 0.63 ± 0.16  6.0 ± 1.0 10.4 ± 0.8 40 1.0 μm Fe(OH)₃(s)4 0.75 ± 0.33  3.3 ± 1.3 24.3 ± 1.6 82  10 μm PCA 3 0.21 ± 0.09  3.3 ±2.1 16.1 ± 4.4 66 1.0 μm Fe(OH)₃(s), 4 4.87 ± 1.32 29.3 ± 8.5 18.9 ± 1.880  10 μm PCA  10 μm PYO 3 9.54 ± 0.18 44.3 ± 5.1 24.1 ± 2.1 94 1.0 μmFe(OH)₃(s), 3 4.20 ± 0.17 31.8 ± 3.5 19.9 ± 2.6 56  10 μm PYO ND = notdetermined. All values are means of results of n images ± standard errorof the mean. Each image covers an area of 3.03 × 10⁵ μm². ¹Calculatedfor the area coverd by biomass.

This rescue occurred even with the total iron concentration (1.2 μM)being more than 10 times lower than that reported in CF sputum(generally greater than 10 μM, ranging from 17 to 200 μM). PCA thereforecan stimulate P. aeruginosa biofilm formation in the presence ofotherwise biologically unavailable Fe(III), even in the absence ofpyoverdin if sufficient iron levels can be attained.

Example 3: PCA Cam Promote Biofilm Formation in the Presence ofConalbumin by Facilitating Fe(II) Uptake

The previous experiments used the effectively insoluble mineralferrihydrite to limit cells for Fe(III). While this may be relevant tounderstanding Fe(III) acquisition by P. aeruginosa in soil environments,in a clinical context, Fe(III) limitation results from binding byhost-produced proteins of the transferrin family (including lactoferrin,serotransferrin, and conalbumin: K_(d)˜10²⁰⁻²³ M⁻¹).

To test whether PCA could stimulate biofilm development by facilitatingFe(II) uptake through reductive liberation of Fe(II) from hostFe(III)-binding proteins, a pathway that is independent ofsiderophore-mediated Fe(III) uptake, biofilm development under a flow of1% TSB was compared between the wild type and the feoB::MAR2xT7 mutant,a strain disrupted in gene PA14_56680, encoding the cytoplasmic membraneprotein FeoB. The identity of this mutant by sequencing and performing adiagnostic phenotypic test, was confirmed as follows.

Because FeoB was recently shown to be the transporter required forenergy-dependent Fe(II) uptake across the cytoplasmic membrane of P.aeruginosa, distinct from the more extensively studied TonB1-dependentABC transport system for siderophore-mediated Fe(III) uptake, it ispredicted that the feoB::MAR2xT7 mutant would not grow on Fe(II). Totest this, experiments were performed in iron-free medium to whicheither Fe(II) or Fe(III) were added back. As expected, the feoB::MAR2xT7mutant could not grow anaerobically in planktonic batch cultures whengiven Fe(II) as its sole iron source, yet could grow in the presence ofFe(III); under these same conditions, the wild type grew regardless ofwhether iron was in the ferric or ferrous form (FIG. 3). In 1% TSBmedium, both strains developed into mature biofilms over time, asexpected (FIG. 4B). However, when the amount of available iron wasreduced by adding the iron chelator, conalbumin (in its iron-free form),neither strain formed biofilms.

Previous researchers have also used conalbumin as a lactoferrinsurrogate in these type of experiments, because they have a similarstructure, Fe(III)-binding capacity, and effect on biofilm formation. Toconfirm the amounts of conalbumin and PCA used in the biofilmexperiments specifically affected the amount of iron required to signalbiofilm formation, and not the amount of iron required for planktonicgrowth, planktonic growth by the wild type and feoB::MAR2xT7 mutant wasmeasured; the additive(s) had no effect on the growth of either strain(FIG. 4A). When both conalbumin and PCA were added, making theacquisition of sufficient iron to signal biofilm formation dependentupon PCA-mediated reduction of conalbumin-bound Fe(III) to Fe(II), thewild type could form biofilms but the feoB::MAR2xT7 mutant could not.

For both the wild type and feoB::MAR2xT7 mutant, in contrast to themature biofilms formed in medium without additives, in medium with addedconalbumin, attached bacteria mostly remained as separated individualcells and failed to form clusters even after 6 days (FIG. 4B). This issimilarly observed for P. aeruginosa strain PAO1 in the presence oflactoferrin. In accord with the conalbumin-induced severe biofilmdefect, little PCA was released into the biofilm effluents by eitherstrain (˜0.05-0.1 μM), as expected for density-dependent phenazineproduction. For the wild type, the addition of PCA to conalbumin-treatedmedium rescued the defect by promoting biofilm growth into a uniformlydistributed lawn type structure within 6 days, distinct from themushroom-like structures observed in control medium (FIG. 4B). WithPCA-induced rescue, by day 6, biofilm biomass increased by a factor of13 compared to biofilms treated with conalbumin alone; the total biofilmbiomass was comparable to that in medium without additives (Table 5).

TABLE 5 Additive(s) to Avg. thickness Max. biofilm PA14 biofilm controlTotal biomass Substratum of biomass thickness Time strain medium (1%TSB) n (μm³/μm²) coverage (%) (μm)¹ (μm) Day 6 WT none 1 7.20 46.7 16.462 40 μg/ml conalbumin 6  0.80 ± 0.08 11.8 ± 1.3  9.0 ± 0.7 32 40 μg/mlconalbumin, 4 10.70 ± 0.49 80.0 ± 4.3 13.2 ± 0.8 36 10 μM PCA 40 μg/mlconalbumin, 1 0.34 4.2 8.7 32 10 μM PYO ND = not determined. All valuesare means of results of n images ± standard error of the mean. Eachimage covers an area of 3.03 × 10⁵ μm². ¹Calculated for the area coverdby biomass.

The wild type biofilm developed similarly with respect to structure andbiomass regardless of whether PCA was added to the control medium,indicating that PCA-induced rescue was specific to the biofilm defectcaused by the conalbumin treatment. On the contrary, for thefeoB::MAR2xT7 mutant with a disrupted Fe(II) transporter, PCA additionfailed to rescue the conalbumin-induced biofilm defect (FIG. 4B).Together, these results indicate that PCA's ability to rescue biofilmformation in the presence of the Fe(III)-binding protein conalbumin isbecause it makes Fe(II) bioavailable by reducing protein-sequesteredFe(III) through extracellular electron transfer.

Example 4: The Phenazine Pyocyanin (PYO) can Affect Biofilm FormationIndependent of Facilitating Fe(II) Uptake

PYO, a well-studied phenazine produced by P. aeruginosa and alsodetected in CF sputum, has been previously shown to affect biofilmformation in LB-based medium. Even though PYO was not released under the1% TSB-based biofilm medium conditions, analogous experiments wereperformed to examine whether PYO might promote biofilm development inthe same manner as PCA. Firstly tested was whether PYO could rescue thebiofilm defect in the PA14 ΔpvdAΔpchE mutant, by comparing the effect ofadding 10 μM PYO alone to the effect of adding 1.0 μM Fe(OH)₃(s) and 10μM PYO together to the base medium (1% TSB). In contrast to PCA's rescueoccurring only together with 1.0 μM Fe(OH)₃(s) addition, PYO rescued thebiofilm defect regardless of whether Fe(OH)₃(s) was present; the rescuewithout Fe(OH)₃(s) was 2 times higher than with Fe(OH)₃(s) addition(FIG. 2, Table 4).

Whether the addition of PYO to conalbumin-treated 1% TSB medium couldrescue the PA14 wild type biofilm defect was tested. Unlike PCA, addingPYO did not rescue the conalbumin-induced biofilm defect (FIG. 4B Table5). This implies that PYO cannot efficiently reduce conalbumin-boundFe(III) under these conditions, which is consistent with PYO being athermodynamically less favorable reductant than PCA. However, thisresult was somewhat unexpected, given an earlier study by Cox reportedthat P. aeruginosa could reduce transferrin-bound Fe(III).

A possible explanation herein provided for guidance purpose and which isnot intended to be limited is that PYO did not stimulateconalbumin-bound Fe(III) reduction for us, but did stimulatetransferrin-bound Fe(II) reduction for Cox, is because one or more ofthe following caveats apply: (i) even though proteins within thetransferrin family (such as conalbumin and transferrin) have similarlystrong Fe(III) binding capacities, the k_(d) values can still bedifferent by 2-3 orders of magnitude [42]; (ii) a given phenazine'sFe(III) reduction activity depends on factors such as concentrationratios between reactants (e.g. phenazine vs. chelated Fe(III)), and/orthe presence of other oxidants that could react more readily withreduced phenazines.

For example, reduced PYO has been shown to be much more reactive towardsoxygen than reduced PCA, making PYO more subject to oxygen competingwith Fe(III) as an electron acceptor [3]. Consistent with this, in thewhole bacterial cell suspension system studied by Cox, PYO wasdemonstrated to be only efficient at reducing transferring-bound Fe(III)under a strict anaerobic condition [43]. Considering our flow cellbiofilm system is very different from the one studied by Cox both withrespect to oxygen content and other factors, it makes sense thatphenazine reactivity would be different. Interestingly, PYO did notstimulate iron-independent rescue under these conditions either, incontrast to what was observed for the ΔpvdAΔpchE mutant under differentiron-limited conditions (FIG. 2).

Example 5: The Effect of Phenazines as a Signaling Compound FacilitatingBacterial Biofilm Development

The Effect of Phenazines on Bacterial Motility.

Previous studies have shown that P. aeruginosa attachment to surfaces ismediated by flagella, whereas movement along colonized surfaces isdriven by pili, giving rise to cell aggregates that grow to form maturebiofilms. P. aeruginosa PA14 is capable of three types of motility: 1)swimming in fluid media, accomplished via reversible rotation offlagella, 2) twitching, a surface-associated movement mediated by typeIV pili, and 3) swarming, which also occurs on solid surfaces and isdependent on flagella. It was found that swimming and twitchingmotilities of the Δphz1/2 mutant were indistinguishable from thewild-type strain. However, swarming motility was significantly higher inΔphz1/2 (FIG. 6). After 40 h incubation, the Δphz1/2 mutant covered onaverage 84% more surface than the wild type. Addition of 100 μM PCA tothe swarming agar had no effect on the wild type, but it significantlydecreased the motility of the Δphz1/2 mutant. Addition of 100 μMpyocyanin had no inhibitory effect on swarming of either the wild typeor Δphz1/2. Altered swarming motility often correlates with differencesin rhamnolipid or other surfactant production. Using a drop-collapseassay significant difference in surfactant production between thewild-type and Δphz1/2 strains was observed.

To measure the impact of phenazines on biofilm formation directly, aflow cell system was first used to grow wild-type and Δphz1/2 biofilms.In this system, the wild type formed heterogeneous biofilms that after 4days developed large and abundant microcolonies (FIG. 7A). Biofilms ofΔphz1/2 were flatter and consisted of fewer and smaller aggregatesscattered throughout the field of view (FIG. 7B). The morphology of4-day-old biofilms was analyzed using COMSTAT. A fixed threshold valueand connected volume filtration were used for all image stacks. Table 6summarizes the values calculated for mean biofilm thickness, substratumcoverage, number of microcolonies at the substratum, surface-to-volumeratio and maximum biofilm thickness for three independent experiments.While the wild type showed a higher maximum biofilm thickness thanΔphz1/2, the phenazine-deficient mutant showed a highersurface-to-volume ratio. When Δphz1/2 biofilms were grown in thepresence of 25 μM pyocyanin (FIG. 7C), larger microcolonies and thickerbiofilms resulted. PCA was not tested under these conditions. The meanthickness, the number of microcolonies and the total biomass volume allincreased, reaching numbers similar or even higher than those observedfor the wild type (Table 6). These results indicate that pyocyaninactively shapes the architecture of P. aeruginosa flow cell biofilms.

TABLE 6 Quantitative analysis of 4 day-old biofilms formed by thewild-type and a phenazine defective mutant. Δphz1/2 Wild-type □Δphz1/2PYO^(a) Total Biomass (μm³/μm²) 2.2 ± 0.49 0.41 ± 0.04 4.3 ± 1   %Coverage at 8 ± 1  2.7 ± 0.6 15.2 ± 2.46 substratum Maximum thickness54.7 ± 6.4  40 ± 2  57.5 ± 1.9  Average thickness 2.4 ± 0.59 0.47 ± 0.035.6 ± 1.6 Number of 9.3 ± 3   2   8 ± 1.5 microcolonies^(b) Average sizeof 46.4 ± 13   12.1 102.6 ± 31   colonies at substratum (μm²) Averagecolony volume 1551 ± 498  251.9 8170 (μm³) Roughness coefficient 1.751.9 1.3 Surface to volume ratio 3.5 ± 0.23 10.5 ± 3.6  6.6 ± 0.6(μm²/μm³) Analysis was conducted using COMSTAT. Images from similarpositions in the flow cell were acquired for all conditions intriplicate. ^(a)25 μM pyocyanin (PYO) was added to the medium from thebeginning of the experiment. ^(b)Minimum microcolony size at thesubstratum was set at 10 μm².

Example 6: The Effect of Phenazines on Colonial Morphology and Structure

To determine whether the morphological trends that were observed in flowcells might translate to a larger scale, colony biofilms of the wildtype and Δphz1/2 were grown. As reported previously, wild type PA14formed smooth colonies that developed concentric ridges only afterprolonged incubation (4 days; FIG. 8A) while Δphz1/2 formed wrinkledcolonies that grew vertically within 2 days. Surface coverage by Δphz1/2colonies was increased by up to 75% compared to wild-type colonies after7 days (FIG. 8B). These features are consistent with an increasedsurface-to-volume ratio in the absence of phenazines, mirroring ourfindings for flow cell biofilms. PCA and pyocyanin were tested for theirspecific effects on surface coverage and rugosity by adding 0.2 M ofeither compound to the agar.

Both phenazines significantly decreased rugosity and surface coverage ofΔphz1/2 colonies, while they did not noticeably affect the structure ofwild-type colonies (FIG. 8A, B). Interestingly, PCA prevented colonyspreading and the formation of wrinkles in Δphz1/2 colonies moreefficiently than pyocyanin. A titration of phenazines showed that 0.1MPCA was sufficient to decrease surface coverage of Δphz1/2 colonies towild-type levels (FIG. 8C), and higher concentrations of PCA (up to0.4M) had no additional effects. In contrast, Δphz1/2 colonies stillshowed 50% increased surface coverage compared to wild-type colonies inthe presence of 0.1 M pyocyanin. At pyocyanin concentrations of 0.2M orhigher, Δphz1/2 covered 35% more of the agar surface than wild-typecolonies. Therefore, the nature of the phenazine, in addition to thetotal amount of phenazine, is an important parameter influencing colonystructure.

Example 7: The Effect of Phenazine in Promoting Survival of BacterialVia Facilitating Intracellular Redox Balancing Ad Homeostasis ThroughCentral Metabolic Pathways

P. aeruginosa PA14 Catalyzes Pyocyanin Reduction.

Stationary-phase LB cultures of P. aeruginosa PA14 turn brightblue-green due to the production of the blue pigment pyocyaninspecifically during this growth phase. P. aeruginosa PA14 also catalyzesthe reduction of pyocyanin, a process that is readily observed when astationary-phase culture is left standing without mixing or aeration bybubbling. Pyocyanin is converted from its blue (oxidized) form to acolorless (reduced) form. At the air-liquid interface, pyocyanin remainsoxidized or becomes re-oxidized by an abiotic reaction with oxygen, butrespiration by the bacteria creates a steep oxygen gradient just belowthis interface such that pyocyanin below a few millimeters remainscolorless. A demonstration of this process is depicted in FIG. 9A (tube3). A stationary-phase culture was centrifuged and the cell pellet wasresuspended in a 100 μM solution of pyocyanin in MOPS buffer, theculture was allowed to sit without shaking for 5 minutes at roomtemperature. A gradient formed that resembled those observed forcultures in growth media. After vortexing, the entire suspensionregained its original blue color (FIG. 9A, tubes 5 and 6). A filtratefrom this suspension had the absorbance spectrum characteristic ofpyocyanin in the oxidation state most stable under atmosphericconditions. When the culture was moved into an anaerobic chamber and astoppered anaerobic cuvette was used to measure the absorbance spectrumof anaerobic culture filtrate, the sample showed decreased absorbance,indicating that pyocyanin had been reduced (FIG. 9B).

Example 8: Pyocyanin Reduction Rates Increase in Stationary Phase

To quantify the rate of pyocyanin reduction by whole cells and testwhether this process, like the biosynthesis of phenazines, was growthphase-dependent, LB culture at different stages of growth was sampled.Samples were diluted into their own supernatant, amended with pyocyanin,and transferred to an anaerobic cuvette. The decrease in oxidizedpyocyanin absorbance was monitored over time for each sample, and amarked increase in the rate of pyocyanin reduction after the appearanceof pyocyanin in stationary phase was observed. This result indicatesthat the rate of pyocyanin reduction by whole cells is growth-phasedependent (FIG. 10).

Example 9: Pyocyanin Exposure Balances the Intracellular Redox State

Strains of P. aeruginosa have been shown to vary in the timing andextent of phenazine production relative to the growth phase. Theappearance of pyocyanin in wild type P. aeruginosa PA14 LB culturescorrelates with entry into stationary phase and pyocyanin productionplateaus in late stationary phase, reaching concentrations ranging from˜100 to 300 μM depending on the growth conditions (FIGS. 11A and 12C).

Given that NADH reacts with pyocyanin in vitro, one potentialconsequence of pyocyanin production and/or exposure would be a decreasein intracellular NADH levels. This is tested by growing cultures of P.aeruginosa wild type and a Δphz mutant (with in-frame deletions of bothphenazine biosynthetic loci) and measuring intracellular NAD(H)approximately four hours after the onset of stationary phase. Theintracellular NADH/NAD⁺ ratio in the wild type was less than half thatobserved for the Δphz mutant. The growth curves for these cultures werevirtually identical under the incubation conditions for this experiment.Addition of 90 μM oxidized pyocyanin (the approximate concentration ofpyocyanin produced by wild type cultures under these conditions) to Δphzmutant cultures reduced the NADH/NAD⁺ ratio to the wild type level (FIG.11B). As a negative control, supernatant from the Δphz mutant wastreated similarly and tested for an effect on intracellular NAD(H)concentrations; no difference was observed between cultures treated with“pyocyanin” preparations from the Δphz mutant and those treated withwater. In titration experiments, an inverse relationship was found toexist between the concentration of pyocyanin added to a Δphz mutantculture and the NADH/NAD⁺ ratio (FIG. 11C).

To test whether the effect of pyocyanin is similar to that of aphysiologically relevant terminal electron acceptor, 30 mM nitrate (aconcentration sufficient to support growth of P. aeruginosa viaanaerobic nitrate respiration) was added to a wild type culture, andnitrate with or without pyocyanin to Δphz mutant cultures in stationaryphase (FIG. 11B). Nitrate and pyocyanin both effected decreases inintracellular NADH/NAD⁺ ratios, apparently by catalyzing NADH oxidation,since decreases in absolute NADH concentrations correlated withincreases in absolute NAD+ concentrations (FIG. 11D). Whereas pyocyanineffected a decrease when added in the micromolar range, nitrate did onlywhen added at millimolar concentrations. Together, these resultssuggested that NADH can act as a source of electrons for pyocyaninreduction.

Example 10: The Intracellular NADH/NA⁺ Ratio is Influenced by theRelative Availability of Electron Donor and Acceptor

The observation that other electron acceptors, i.e., pyocyanin andnitrate, decreased the NADH/NAD⁺ ratio suggested that oxygen waslimiting during stationary phase in the cultures. This could explain theaccumulation of NADH four hours after the onset of stationary phase inthe Δphz mutant (FIG. 11B). To confirm this, a batch culture of the Δphzmutant was grown in a fermentor, which allowed temperature and aerationcontrolling while simultaneously measuring dissolved oxygen in theculture. The culture was sampled at regular intervals to measure opticaldensity and extract NAD(H). As predicted, oxygen levels decreased slowlyuntil the culture reached mid- to late exponential phase, at which timeit plummeted to zero. This drop in oxygen correlated with an increase inthe intracellular NADH/NAD⁺ ratio (FIG. 12A).

To test whether the drop in oxygen depended on the availability ofelectron donors for oxygen reduction, the experiment was repeated withadded 20% of the glucose concentration in the medium compared to themedium in the initial experiment (10 mM versus 50 mM). When lesselectron donor was available, the oxygen concentration decreased inmid-exponential phase, but never reached zero and rapidly increasedagain upon entry into stationary phase (FIG. 12B). This culture neverreached the same growth yield achieved by the culture containing 50 mMglucose, implying that the carbon source was the limiting factor thatled it to enter stationary phase. The culture experienced oxygenlimitation only transiently, if at all, due to the lower ratio ofelectron donor to electron acceptor in the experiment depicted in FIG.12B compared to FIG. 12A. As a result, the NADH/NAD⁺ ratio never reachedthe high level observed for the culture containing excess glucose.

Finally, the wild type strain in the presence of 50 mM glucose wastested, and sampled for pyocyanin concentrations in addition to NAD(H)and cell density. The wild type strain also exhibited increasedNADH/NAD⁺ ratios upon entry into stationary phase, and these ratioscorrelated with oxygen limitation. However, unlike the Δphz mutant, thewild type showed a decrease in intracellular NADH/NAD⁺ that correlatedwith the appearance of pyocyanin in the culture. These results furthersupport the hypothesis that pyocyanin can act as an alternate oxidantunder conditions where the terminal electron acceptor for respirationhas become limiting. This interpretation derives from the largedifference in NADH levels observed between the wild type strain and Δphzafter about 12 hours of incubation, and the correlation betweendecreasing NADH levels and increasing pyocyanin concentrations inculture filtrates observed upon entry into stationary phase (FIG. 12C).

Example 11: P. aeruginosa PA14 Excretes, and them Consumes, Pyruvate inLate Stationary Phase

For fermentative organisms such as E. coli and Propionibacteriumfreudenreichii, the addition of the synthetic redox-cycling compoundferricyanide has been shown to alter carbon flux through centralmetabolic pathways. Particularly when the re-oxidation of this compoundis coupled to electron transfer to an electrode, ferricyanide shiftedthe fermentation balance away from ethanol and propionate, products thatrequire NADH for their formation, toward acetate, a more oxidizedproduct. This implies that the ferricyanide acts as an electron shuttlefrom major pools of reductant inside the cell, such as NADH, to theelectrode, thereby lessening the need for formation of more reducedfermentation products to dissipate cellular reductant.

To determine whether pyocyanin could play a similar role in P.aeruginosa, filtered culture supernatants was analyzed for small organicacids that are known fermentation products of P. aeruginosa metabolism.P. aeruginosa has been shown to ferment pyruvate under energy-starvedconditions, converting it to lactate, acetate, and/or succinate. Theproduction of lactate or succinate from pyruvate requires NADH as asubstrate, while the conversion of pyruvate to acetate requires NAD⁺.Therefore, the NADH/NAD⁺ ratio in the wild type would be more favorablefor acetate production, whereas the NADH/NAD⁺ ratio in the Δphz mutantwould favor production of lactate and succinate.

Surprisingly, a marked difference was observed between the wild type andthe Δphz mutant with respect to the production of pyruvate itself. Inlate stationary-phase (about 30 hours after inoculation) after growth ina defined medium with 50 mM glucose, pyruvate concentrations wereobserved as high as 6 mM in wild type culture filtrates (as indicated bya peak eluting at about 10.5 minutes), but any pyruvate in filtratesfrom Δphz mutant cultures was unable to detect. Adding pyocyanin to theΔphz mutant upon entry into stationary phase complemented the pyruvateexcretion phenotype (FIG. 13), although incompletely because only abouthalf the final concentration of pyocyanin produced by the wild typeunder these conditions was added (50 vs. 100 μM). Citrate, lactate andacetate in both wild type and Δphz mutant culture filtrates were alsodetected at similar concentrations, eluting at ˜9.1, 14.3 and 17.0minutes, respectively. The peak eluting at 7.1 minutes was the MOPSbuffer from the medium. The compounds represented by the peaks elutingat approximately 7.3 minutes (wild type filtrate only), and 9.9 and 12.2minutes (both wild type and Δphz mutant filtrates) were not identified.Standards containing 2-oxoglutarate and malate were run with the samemethod, but did not co-elute with any of these peaks.

To better constrain the timing of metabolite excretion in the wild typeand Δphz mutant, duplicate cultures were sampled every 4 hours over thecourse of approximately 30 hours in stationary phase (FIG. 14). Pyruvateappeared at detectable levels in wild type cultures between 22 and 26hours after inoculation, and had increased to ˜5 mM after 38 hours.However, by the 42-hour time point, the pyruvate in both replicates haddecreased to levels below the detection limit (˜0.05 mM) (FIG. 14C).Abiotic degradation of pyruvate generates a peak eluting atapproximately 8 minutes, which does not co-elute with any of the peaksobserved in traces from our culture filtrates (data not shown).Therefore, the disappearance of the pyruvate peak at the 42-hour timepoint implied that it had been metabolized by the bacteria.

Another phenotype that became apparent under these growth conditions wasthe reproducible difference in cell yields between wild type and Δphzmutant cultures. The optical densities of wild type cultures weretypically lower than those of the Δphz mutant cultures in stationaryphase, a phenotype that becomes more apparent when the optical densityis plotted on a linear scale (FIGS. 14A and 14B).

Example 12: Pyruvate Fermentation Facilitates Survival is Energy-StarvedP. aeruginosa PA14 Cultures

Recently, Schobert and colleagues have characterized genes implicated ina pyruvate fermentation pathway in P. aeruginosa strain PAO1. In thispathway, pyruvate is converted by multiple enzymes to succinate,acetate, and/or lactate. These reactions were probably responsible forthe consumption of pyruvate in late stationary phase in the cultures,because these compounds are detectable by the analytical HPLC method,and their concentrations increase was not observed as pyruvate.Therefore, it is hypothesize that pyruvate was completely oxidizedthrough the utilization of the small amount of oxygen available to thecells. However, in environments with steep gradients of electronacceptor availability, such as those encountered in surface-attached oraggregated bacterial communities, excreted pyruvate may be utilized forsubstrate-level phosphorylation when respiratory electron acceptorsbecome limiting. To verify that P. aeruginosa strain PA14 can utilizepyruvate for survival under strict anaerobic conditions, the wild typeand an IdhA mutant, defective in the ability to reduce pyruvate tolactate, were incubated in stoppered serum bottles containing bufferedLB amended with 20 mM pyruvate. Wild type culture with no pyruvate wasset up as a control. Colony-forming units in samples from these cultureswere monitored over more than three weeks, and showed that, a mutantwith a disruption in the gene IdhA was defective in survival onpyruvate. The decline of this mutant was similar to that of the wildtype culture containing no added pyruvate. P. aeruginosa PA14 istherefore also able to survive under conditions of energy starvationthrough utilization of a lactate dehydrogenase-dependent pathway forpyruvate fermentation.

Example 13 A Model for Pyocyanin Reduction Allows P. aeruginosa PA14 toMaintain Redox Homeostasis Under Oxygen-Limited Conditions

When sufficient oxygen is available for growth (FIG. 15A), the aerobicrespiratory chain (“resp”) can catalyze the reoxidation of NADH. Underconditions in which terminal electron acceptors for respiration arelimiting (FIG. 15B), P. aeruginosa can couple the reoxidation of NADH tothe reduction of pyocyanin, either directly or through anenzyme-mediated reaction as represented by “pyocyanin red,” a putativephenazine reductase. The electrons could be transferred from pyocyaninto oxygen through an abiotic extracellular reaction. (FIG. 15C) Alsounder conditions of oxygen limitation, the NADH/NAD⁺ ratio could bebalanced through inactivation of the pyruvate dehydrogenase complex bypyocyanin. NAD⁺ reduction (and therefore NADH production) would beavoided because pyruvate would be excreted without further oxidation.

Example 14: The Effect of Phenazine in Promoting Anaerobic Survival ofBacteria Under Conditions of Oxidant Limitation

Antibiotics are increasingly recognized as having other, importantphysiological functions for the cells that produce them. An example ofthis is the effect phenazines have on signaling and communitydevelopment for Pseudomonas aeruginosa. Phenazine-facilitated electrontransfer to poised-potential electrodes promotes anaerobic survival butnot growth of Pseudomonas aeruginosa PA14 under conditions of oxidantlimitation. Other electron shuttles that are reduced but not made byPA14 do not facilitate survival, suggesting the survival effect isspecific to endogenous phenazines. Examples are shown in the enclosedpaper: Wang et al., 2010, Endogenous phenazine antibiotics promoteanaerobic survival of Pseudomonas aeruginosa via extracellular electrontransfer (J. Bacteriology, vol. 192, No. 1, page 365-369).

Examples 15: Identification of Phenazine-Degrading Bacterial Strainsthat Produce Phenazine Degrading Enzymes

Bacteria that degrade phenazines has been isolated and identified. Forexample, a new phenazine-degrading strain was isolated that preliminary16S rDNA sequencing results suggest is closely related to Mycobacteriaand Streptomyces species. This bacterium was isolated on minimal mediumusing PCA as the sole carbon source. The relationship between bacteriumgrowth and PCA degradation suggested that the bacterial strain could usephenazines as the sole source of carbon and nitrogen and was able tocompletely degrade PCA in a short time.

Isolation of additional novel phenazine degraders is expected to beperformable by using a similar method: construct an “enrichment culture”by defining a minimal growth medium where a phenazine (PCA, PYO, etc. .. . ) is provided as either (or both) the sole source of carbon ornitrogen. If growth is observed after many rounds of serial dilutions,phenazine-degraders can be isolated by plating the enrichment culture onan agar plate with the same medium composition. Single colonies arepicked, and streaked on fresh plates, and visually checked for purity.Once pure, the 16S rDNA is sequenced and the organism can bephenotypically characterized.

Another example of phenazine degrading bacterial strains is provided inthe paper: Yang, Z-J et al., 2007, “Isolation, identification, anddegradation characteristics of phenazine-1-carboxylic acid-degradingstrain Sphingomonas sp. DP58”. Current Microbiology. 55:284-287, hereinalso incorporated by reference in its entirety.

Example 16: Correlation Between Elevated Phenazine Concentrations andthe Decline of Pulmonary Function

To determine the relationship of FEV1% to phenazine concentrations,forty-seven participants were recruited during scheduled visits toChildren's Hospital of Boston (CHB). Inclusion criteria were a diagnosisof CF based on genotyping and sweat chloride testing and chronic P.aeruginosa infection (positive culture>1 year). Sputum samples wereobtained from each patient by expectoration. Samples were stored on iceand processed within 4 hours of expectoration. Sputum was shearedthrough a syringe and homogenized in an equal volume of 1 mM Sputolysinfor 30 minutes.

Homogenized sputum was centrifuged and supernatants were filteredthrough filter centrifuge columns and phenazine content was quantifiedby HPLC as described in Dietrich et al (Molecular Microbiology, 2006.61(5): p. 1308-1321).

FEV1% values were determined by spirometry as described in Knudson etal., Am Rev Respir Dis 1976; 113:587-600. In particular, a crosssectional analysis of sputum pyocyanin concentrations reveals a positivecorrelation with pulmonary function decline (FEV1%) as illustrated inFIG. 16A.

One method to detect whether Fe(II)/Fe(III) chelation and/or inhibitionof phenazine-mediated iron acquisition is successful, is to use a directimaging approach. For example, the hybridization chain reaction (Choi etal., “Programmable in situ amplification for multiplexed imaging of mRNAexpression” Nature Biotechnol., 2010) can be used to detect geneexpression at high resolution at the single cell level.

As an example, using genes such as bqsS and bqsR (Kreamer et al., J.Bacteriol, 2011, 194, 1195-1204), which encode a two-component signaltransduction system that is upregulated specifically in response toferrous iron [Fe(II)], detecting the expression of these genes (or lackthereof) within lung samples, including expectorated sputum, could beused as a direct indicator of the inhibition of phenazine-mediatedpathways, including iron acquisition.

Reference is made to hybridization chain reaction imaging of wild typeP. aeruginosa and a mutant containing a deletion of bqsR, illustrated inFIG. 16B and FIG. 16C. The result reveal differential gene expressionpatterns of the genes bqsS (green) and bqsR (red) in response to 50 μMFe(II) (left) and no treatment (right) (FIG. 16B and FIG. 16C)

Example 17: The Phenazine Pyocyanin is a Terminal Signaling Factor inthe Quorum Sensing Network of Pseudomonas aeruginosa

Using Pseudomonas aeruginosa DNA microarrays and quantitative RT-PCR, itis demonstrated that the phenazine pyocyanin elicits the upregulation ofgenes/operons that function in transport [such as theresistance-nodulation-cell division(RND) efflux pump MexGHI-OpmD] andpossibly in redox control (such as PA2274, a putative flavin-dependantmonooxygenase), and downregulates genes involved in ferric ironacquisition. Strikingly, mexGHI-opmD and PA2274 were previously shown tobe regulated by the PA14 quorum sensing network that controls theproduction of virulence factors (including phenazines). Throughmutational analysis, it is shown that pyocyanin is the physiologicalsignal for the upregulation of these quorum sensing controlled genesduring stationary phase and that the response is mediated by thetranscription factor SoxR. Our results implicate phenazines as signalingmolecules in both P. aeruginosa PA14 and PAO1. Examples are shown in thepaper: Dietrich et al. 2006, The phenazine pyocyanin is a terminalsignaling factor in the quorum sensing network of Pseudomonasaeruginosa, Molecular Microbiology Vol. 61, No. 5, page 1308-1321,incorporated herein by reference in its entirety.

Example 18: PCA Degradation Using Isolate DKN1213

Soil samples were inoculated into a minimal salts liquid medium with ˜5mM PCA provided as the sole carbon source. After 1 week of incubation at30 C, a 10% inoculum was introduced into a fresh batch of the samemedium with PCA again serving as the carbon source. After an additionalround of enrichment, 100 uL of the enrichment culture were spread uponan agar petri dish made of the same medium+5 mM PCA. After a week'stime, single colonies were picked and re-streaked to the same agarmedium for purification, as well as to an LB plate. After several morerounds of streaking to verify purify, a single colony was picked andgrown up in minimal medium+PCA. This sample was cryopreserved, andbecame the standard reference culture for later experiments (namedDKN1213).

16s rRNA sequencing was performed on DKN1213, revealing it to be closelyrelated to strains of fast growing Mycobacteria, as well as toStreptomyces. When DKN1213 was grown in LB broth+4 mM PCA, degradationof over 1 mM PCA was measured in less than a week, indicating that itcontains an enzyme capable of degrading PCA, as well as a system fortransporting PCA into the cell. Similar enrichment and isolation methodsare expected to be suitable to enrich other novel forms of PCAdegraders. Instead of providing PCA as the carbon source, PCA can beprovided as the nitrogen source, or as both the carbon and nitrogensource. Analogously, other phenazines are expected to be used to enrichfor phenazine-degrading bacteria in the same way as described for PCA.

Further work to identify the enzyme responsible for degrading PCA can beperformed with DKN1213. A biochemical approach is expected to requirethat an activity assay be developed for PCA degradation. This isexpected to be based on following either the absorption or fluorescenceof PCA over time, and purifying cell fractions that promote itsdisappearance. A genetic approach is expected to employ transposonmutagenesis to make a collection of random mutants and screen them forthe inability to grow on a minimal medium+PCA. Once an enzyme isidentified, its specificity can be altered using directed evolution, soas to change the spectrum of phenazines it could recognize or improveits efficiency.

Example 19: Fe(II)/Fe(III) Combination Therapy for Cystic FibrosisPatients

The abundance of Fe(II) in the lungs of cystic fibrosis patients hasimportant implications for the design of novel antimicrobial therapies.

Competition between pathogens and their hosts for ferric iron [Fe(III)]has been extensively studied due to iron's critical importance inpathogenesis [44]. While microbial ferrous iron [Fe(II)] uptake pathwaysare known [45], therapeutic strategies designed to limit ironavailability have only targeted Fe(III) because it is commonly assumedto be the dominant physiologically relevant form.

For example, Fe(III) chelation has been shown to dramatically improveantibiotic effectiveness against the opportunistic pathogen Pseudomonasaeruginosa in aerobic environments, and is being explored as a means tocombat biofilm infections of cystic fibrosis (CF) patients [46-49].Based on the results obtained by this approach in vitro, it is expectedthat if it is to be similarly effective in vivo, iron would need toremain in its oxidized state [Fe(III)] as infections progress.

However, in late stages of CF infections, localized hypoxicmicroenvironments exist [50] which could stabilize Fe(II). Furthermore,P. aeruginosa produces redox-active phenazines in CF sputum [51] thatcan reduce Fe(III) to Fe(II) and circumvent Fe(III)-chelation in vitro[52].

Ferric iron [Fe(III)] chelation has been shown to combat pathogenicmicrobial biofilms in vitro, and has been proposed as a novel treatmentfor cystic fibrosis (CF) patients. However, the success of this approachassumes an abundance of Fe(III) in the infected environment. Here weshow that appreciable levels of ferrous iron [Fe(II)] exist in themajority of CF lungs, that Fe(II) compromises Fe(III) chelation therapyunder anaerobic conditions, and that Fe(III) and Fe(II) chelators canact synergistically to prevent or disrupt biofilm growth.

In particular, Examples 20-22 (below) show that Fe(II) can be abundantat infection sites, and its concentration was measured in CF sputum frompatients at different stages of lung function decline. While total ironhas been quantified previously [53], this is the first report of itsoxidation state in vivo.

Example 20: Demonstration of the Presence of Fe(II) in Sputum of CysticFibrosis Patients

Twenty-five participants with cystic fibrosis (CF), aged 7 to 20, wererecruited during scheduled visits to Children's Hospital Los Angeles(CHLA). Study inclusion criteria were a positive diagnosis of CF,ability to expectorate sputum and informed consent/assent. Diseaseseverity was determined by FEV1% scores using published guidelines [55].CHLA and the California Institute of Technology approved the studyprotocols (CCI-10-00232).

A total of 116 sputum samples from 25 patients were immediately flashfrozen upon expectoration and moved to an anaerobic chamber foranalysis. Samples were homogenized and ratios of Fe(II)/Fe(III)concentrations were determined using the Ferrozine® assay. Total ironlevels were confirmed using ICP-MS and increased significantly as lungfunction worsened (FIG. 17 Panel A). In most patients, a notableproportion of total iron was Fe(II) (>19%), though it was appreciablyhigher (>37%) in subjects with mild to severe pulmonary obstruction.That such high concentrations of Fe(II) are observed at all stages ofinfection is striking, and reinforces the need to better understand themechanisms of iron homeostasis in the lung environment [54].

Example 21: Demonstration of Synergistic Effect of Fe(II) and Fe(III)Chelators on Bacterial Biofilms

Because the abundance of Fe(II) in infected environments of lungs of CFpatients may compromise the success of Fe(III)-specific chelationtherapies, the question of whether a combination of Fe(III) and Fe(II)chelators would be more effective than Fe(III) chelators alone wasinvestigated. An anaerobic, high-throughput biofilm assay was used todetermine whether Ferrozine®, an Fe(II)-specific chelator, could actsynergistically with conalbumin, an Fe(III) chelator, to prevent P.aeruginosa biofilm formation (FIG. 17 Panel B). Neither compoundaffected planktonic growth rates. Contrary to aerobic observations[46,47], 100 μM conalbumin was also ineffective in preventing biofilmgrowth under anaerobic conditions where ˜10 μM Fe(II) and 10 μM Fe(III)were present. In contrast, 100 μM Ferrozine® reduced biofilmaccumulation by 24% and 200 μM Ferrozine® reduced it slightly further.Strikingly, the combination of 100 μM conalbumin and 200 μM Ferrozine®reduced biofilm accumulation by 54%. To determine whether this effectwas due to iron sequestration rather than non-specific interactions,Fe(II) in excess of the chelation capacity was added. Under theseconditions, biofilm growth was restored. Iron not only signals biofilmformation, but is involved in biofilm maintenance [49].

Example 22: Demonstration of Synergistic Effect of Fe(II) and Fe(III)Chelators on Mature Bacterial Biofilms

Because iron not only signals biofilm formation, but is involved inbiofilm maintenance [49], similar mixed Fe(II)/Fe(III) chelationexperiments targeting mature biofilms (FIG. 17 Panel B) were performed.Conalbumin did not significantly reduce established biomass, but ˜20%dissolution was observed in the presence of 100 or 200 μM Ferrozine®.Together with conalbumin, Ferrozine® promoted even more dissolution, yetbiomass was maintained at high levels in the presence of excess Fe(II).Collectively, the results of Examples 20-22 indicate that as lungfunction declines and Fe(II) concentrations rise, targeting bothoxidation states will be more effective than targeting Fe(III) alone.

These results highlight that the chemistry of infected environments canbe used gain a clear picture of what pathogens are experiencing. Thiscan be particularly important because pathways that may be crucial forsurvival in vivo may not be the same as those required for survivalunder standard laboratory conditions. Because the environment ofinfections is dynamically responsive to changes in both the microbialcommunity and the host, and is likely heterogeneous on the microscale, amore thorough understanding of its composition in time and space caninform the design of therapeutics. These results demonstrate thepotential for environmentally-informed rational drug design.

In summary, in several embodiments provided herein are methods andsystems for interfering with viability of bacteria and related compoundsand compositions. In particular, in view of above, interfering withviability of bacteria can be performed in vivo or in vitro byinactivating a phenazine and/or one or more phenazine related pathwaysin the bacteria to reduce survivability and/or antibiotic resistance ofthe bacteria according to several approaches as will be understood by askilled person.

According to a first approach, inactivating a phenazine and/or one ormore phenazine related pathway can be performed by inhibiting synthesisof the phenazine in the bacteria. In particular according to the firstapproach the inhibiting can be performed by interfering with quorumsensing of the bacteria, by reducing the amount of phenazine in thebacteria and/or by inhibiting transcription and/or translation of thephenazine biosynthetic genes. According to the first approach theinhibiting and/or the reducing can be performed by inactivating one ormore proteins involved in phenazine biosynthesis which can in particularcomprise acyl homoserine lactones and the pseudomonas quinolone signal(PQS). According to the first approach, the reducing can be performed byenhancing phenazine degradation endogenously and/or exogenously and/orby modifying the phenazines, for example chemically, to inhibit and inparticular prevent phenazine uptake by the bacteria. According to thefirst approach enhancing phenazine degradation can be performed byexpressing and/or delivering a protein that degrades phenazines.

According to a second approach which can be performed in addition or inalternative with the first approach, inactivating a phenazine and/or oneor more phenazine related pathway can be performed by inhibitingtransportation of phenazines in and/or out of the bacterial cell.According to the second approach, the inhibiting can be performed byinhibiting and in particular, blocking one or more phenazine exportersof the bacteria, such as RND efflux pumps of the mexGHIopmD variety andwhen the bacterium is Pseudomonas aeruginosa, one or more phenazineexporters are encoded by PA4205, PA 4206, PA 4207 and/or PA4208.According to the second approach, the inhibiting can also beadditionally or alternatively performed by inhibiting an in particularblocking a protein involved in modifying phenazines in a phenazinemodified form to be recognized by a phenazine exporter of the bacteria.According to the second approach, the inhibiting can also beadditionally or alternatively performed by inhibiting is performed byinhibiting and in particular blocking one or more MFS transportersinvolved in phenazine import/export of the bacteria, such as when thebacterium is Pseudomonas. aeruginosa, one or more MFS transporters areencoded for example by PA3718 and/or PA4233.

According to a third approach which can be performed in addition or inalternative with the first approach and/or second approach, inactivatinga phenazine and/or one or more phenazine related pathway can beperformed by providing the bacteria with one or more phenazine degradingenzymes, such as phenazine degrading enzymes produced byphenazine-degrading bacteria strains and additional enzymes andcompounds identifiable by a skilled person.

According to a fourth approach which can be performed in addition or inalternative with the first approach, second approach and/or thirdapproach, inactivating a phenazine and/or one or more phenazine relatedpathway can be performed by converting at least a portion of thephenazine of the bacteria in an inactive form. According to the fourthapproach converting at least a portion of the phenazine of the bacteriain an inactive form can be performed by inhibiting intracellularreduction and/or extracellular oxidation of phenazines of the bacteria,and/or by modifying phenazines chemically to interfere with phenazineuptake and/or intracellular processing of bacteria.

In any one of the first, second, third and fourth approaches the one ormore phenazine related pathways can comprise phenazine-mediatedbacterial biofilm development in the bacteria and/or phenazine-mediatediron acquisition of bacteria. In particular, in any one of the first,second, third and fourth approaches inactivating phenazine-mediated ironacquisition of bacteria can be performed by inhibitingphenazine-mediated Fe (III) reduction to Fe (II), and/or by inhibitingFe (II) acquisition of bacteria In any one of the first, second, thirdand fourth approaches, inhibiting Fe (II) acquisition of bacteria can beperformed by inhibiting a cytoplasmic membrane Fe (II) transporter ofbacteria, such as the Fe (II) transporter is the cytoplasmic membraneprotein FeoB or a homologues protein thereof. In any one of the first,second, third and fourth approaches inactivating phenazine-mediated ironacquisition of bacteria can be performed by activating a Fe (II)chelator in the bacteria, and in particular a Fe (II) chelator in theform of a protein and/or a chemical compound. In any one of the first,second, third and fourth approaches the Fe (II) chelator can beFerrozine®, and the activating can be performed for example bydelivering Ferrozine® into the mucus environment of bacteria using anaerosol. In any one of the first, second, third and fourth approachesthe Fe (II) chelator is a host protein activating a Fe (II) chelatorcomprises regulating of one or more host genes encoding a host Fe (II)chelator.

In any one of the first, second, third and fourth approaches the one ormore phenazine related pathways can comprise a phenazine-mediatedsignaling pathway of the bacteria, such as a signaling pathway triggersa transition from the motile to the sessile state in bacteria having amotile and a sessile state. In any one of the first, second, third andfourth approaches, inactivating a phenazine-mediated signaling pathwaycan be performed by inactivating one or more signaling molecules (e.g.one or more proteins) in the phenazine mediated signaling pathway, andin particular by inactivating direct or indirect effectors of phenazinesin the pathway, acyl homoserine lactones and/or the pseudomonasquinalone signal (PQS). In any one of the first, second, third andfourth approaches inactivating the one or more signaling molecules isperformed by inhibiting expression of one or more genes in the bacteriacoding for signaling molecules in the pathway.

In any one of the first, second, third and fourth approaches the one ormore phenazine related pathways comprise phenazines related pathwaysforming central metabolic pathways of the bacteria.

In any one of the first, second, third and fourth approaches the one ormore phenazine related pathways comprise intracellular phenazinemediated redox hemostasis of the bacteria, e.g. by inhibitingphenazine-mediated electron shuttling of the bacteria.

In any one of the first, second, third and fourth approaches the one ormore phenazine related pathways comprise transportation of phenazines inand/or out of the bacterial cell.

In any one of the first, second, third and fourth approaches thebacterium is Pseudomonas aeruginosa.

In any one of the first, second, third, and fourth approaches, theapproach further comprises degrading phenazines in vivo and/or in vitro.

In any one of the first, second, third and fourth approaches a systemfor interfering with viability of bacteria according to the approach cancomprise one or more agents able to inactivate a phenazine and/or aphenazine related pathway in the bacteria for simultaneous combined orsequential use in any one of the methods and approaches hereindescribed, optionally together with an antibiotic and/or otherantimicrobial.

According to a fifth approach, inactivating a phenazine and/or one ormore phenazine related pathway can be performed in a method for treatingand/or preventing a bacterial infection in an individual, the methodcomprising administering an effective amount of one or more agents ableto specifically inactivate phenazine and/or a phenazine related pathwayin the bacteria optionally together with administering an antibioticand/or an additional antimicrobial to the individual. According thefifth approach inactivation of phenazine can be performed according toany one of the first, second, third and fourth approached whereininactivation is selective to the phenazine and/or the one or morephenazine related pathways of the bacteria. According to the fifthapproach a system for treating and/or preventing a bacterial infectionin an individual, can comprise one or more agents able to selectivelyinactivate a phenazine and/or a phenazine related pathway in thebacteria and optionally an antibiotic and/or other antimicrobial, forsimultaneous combined or sequential use in any one of the methodsaccording to the fifth approach.

According to a sixth approach, inactivating a phenazine and/or one ormore phenazine related pathway can be performed in a method foridentifying an antimicrobial, comprising contacting a microbe with acandidate agent and detecting the ability of the candidate agent ofinactivating a phenazine and/or a phenazine related pathway in thebacteria. According to the sixth approach, the method can furthercomprise contacting the microbe with an antibiotic and/or an additionalantimicrobial to the individual. According to the sixth approach,inactivation of phenazine is performed according to any one of thefirst, second, third, fourth or fifth approaches wherein inactivation isselective to the phenazine and/or the one or more phenazine relatedpathways of the bacteria. According to the sixth approach, a system foridentifying an antimicrobial, can comprise one or more microbe and oneor more agents capable of detecting phenazine and/or phenazine relatedpathways for simultaneous combined or sequential use in methods andsystems according to any one of the first, second, third, fourth andfifth approach.

According to a seventh approach, inactivating a phenazine and/or one ormore phenazine related pathway can be performed by an antimicrobialcomprising one or more agents able to inactivate a phenazine and/or aphenazine related pathway in the bacteria to reduce antibioticresistance and/or survivability of bacteria and optionally a compatiblevehicle for effective administrating and/or delivering of the one ormore agents to an individual. In particular, according to the seventhapproach, the one or more agents are agents capable of interfering withviability of bacteria in the method and systems according to any one ofthe first, second, third, fourth and fifth approach. According to theseventh approach, the antimicrobial can further comprise an antibioticand/or an additional antimicrobial. According to the seventh approach,the vehicle can be a pharmaceutically acceptable vehicle and thecomposition is a pharmaceutical composition.

According to an eighth approach, inactivating a phenazine and/or one ormore phenazine related pathway can be performed in a method toinactivate a bacterium in vitro or in vivo, the method comprisingcontacting the bacterium with one or more agents capable of inactivatinga phenazine and/or a phenazine related pathway in the bacterium incombination with one or more antibiotic and/or other antimicrobial.According to the eighth approach, the one or more agents are agentscapable of interfering with viability of bacteria in the method andsystems according to any one of the first, second, third, fourth andfifth approach. According to the eighth approach, the one or more agentscan be able to selectively inactivate the phenazine and/or phenazinerelated pathway in the bacterium. According to the eighth approach, asystem for inactivating a bacterium in vitro or in vivo, can compriseone or more agents able to selectively inactivate a phenazine and/or aphenazine related pathway in the bacteria and optionally an antibioticand/or other antimicrobial, for simultaneous combined or sequential usein the method and systems according to any one of the first, second,third, fourth and fifth approach.

According to a ninth approach, inactivating a phenazine and/or one ormore phenazine related pathway can be performed in a method forinterfering with viability of bacteria in a mucous environment, themethod comprising: activating an Fe (II) chelator in the bacteria, theactivating being performed by delivering the Fe(II) chelator into amucus environment. According to the ninth approach, the delivering ofthe Fe(II) chelator into a mucus environment can be performed using anaerosol comprising the Fe(II) chelator with the Fe(II) chelator possiblybeing an Fe(II)-chelating protein or Fe(II)-chelating compound.According to the ninth approach the Fe(II) chelator can be a conalbumin.

According to a tenth approach, inactivating a phenazine and/or one ormore phenazine related pathway can be performed in a method forinterfering with viability of bacteria, the method comprising:activating an Fe(III) chelator in the bacteria, and activating an Fe(II)chelator in the bacteria. According to the tenth approach, the Fe(III)chelator and the Fe(II) chelator are administered for a time and undercondition to substantially prevents and/or disrupts biofilm growth.According to the tenth approach, the Fe(III) and Fe(II) chelators can beadministered for a time and under condition to act synergistically tosubstantially prevent and/or disrupt biofilm growth. According to thetenth approach, the Fe(III) and Fe(II) chelators can be administered fora time and under condition to disrupt mature biofilms.

According to an eleventh approach, inactivating a phenazine and/or oneor more phenazine related pathway can be performed in a method fortreating cystic fibrosis comprising: administering a therapeuticallyeffective amount of a composition comprising an Fe(II) chelator alone orin combination with and an Fe(III) chelator to a individual. Accordingto the eleventh approach, the administering can be performed by way ofan aerosol comprising the Fe(III) chelator and/or the Fe(II) chelator.According to the eleventh approach, the Fe(II) chelator can beFerrozine®, and an amount Ferrozine® and the therapeutically effectiveamount of the composition can range from 10-1000 μM. According to theeleventh approach, the Fe(II) chelator can be conalbumin, and thetherapeutically effective amount of the composition can range from10-1000 μM.

According to a twelfth approach, inactivating a phenazine and/or one ormore phenazine related pathway can be performed by a compositioncomprising one or more agents suitable to inactivate a phenazine and/orone or more phenazine related pathways in a bacteria. According to thetwelfth approach, the one or more agents can comprise an Fe(II) chelatorand/or an Fe(III) chelator and in particular Ferrozine®, possiblycomprised in the composition in an amount ranging between 10-1000 μM,and/or conalbumin, possibly comprised in the composition in an amountranging between 10-1000 μM. According to the twelfth approach, thecomposition can be formulated to reduce biofilm accumulation by greaterthan approximately 50%. According to the twelfth approach, thecomposition can be a pharmaceutical composition in for treatment ofcystic fibrosis and possibly further comprise a suitable vehicle foradministering and/or delivering the one or more agents to an individual.According to the twelfth approach, the composition can be formulated fortopical administration and in particular being in the form of aerosol.

In several embodiments provided herein, methods and systems forinterfering with viability of bacteria and related compounds andcompositions can also be performed by subtracting Fe(II) from the mediumpossibly in combination with subtracting Fe(III). Fe(II) subtraction canbe performed in combination with inactivating a phenazine and/or one ormore phenazine related pathway according with any one of the first totwelfth approaches herein described.

The examples set forth above are provided to give those of ordinaryskill in the art a complete disclosure and description of how to makeand use the embodiments of the compounds, compositions, systems andmethods of the disclosure, and are not intended to limit the scope ofwhat the inventors regard as their disclosure. All patents andpublications mentioned in the specification are indicative of the levelsof skill of those skilled in the art to which the disclosure pertains.

The entire disclosure of each document cited (including patents, patentapplications, journal articles, abstracts, laboratory manuals, books, orother disclosures) in the Background, Summary, Detailed Description, andExamples is hereby incorporated herein by reference. All referencescited in this disclosure are incorporated by reference to the sameextent as if each reference had been incorporated by reference in itsentirety individually. However, if any inconsistency arises between acited reference and the present disclosure, the present disclosure takesprecedence. Further, the computer readable form of the sequence listingof the ASCII text file P756-USC-Sequence-Listing-ST25 is incorporatedherein by reference in its entirety.

The terms and expressions which have been employed herein are used asterms of description and not of limitation, and there is no intention inthe use of such terms and expressions of excluding any equivalents ofthe features shown and described or portions thereof, but it isrecognized that various modifications are possible within the scope ofthe disclosure claimed. Thus, it should be understood that although thedisclosure has been specifically disclosed by preferred embodiments,exemplary embodiments and optional features, modification and variationof the concepts herein disclosed can be resorted to by those skilled inthe art upon the reading of the present disclosure, and that suchmodifications and variations are considered to be within the scope ofthis disclosure as defined by the appended claims.

It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting. As used in this specification and the appended claims,the singular forms “a,” “an,” and “the” include plural referents unlessthe content clearly dictates otherwise. The term “plurality” includestwo or more referents unless the content clearly dictates otherwise.Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the disclosure pertains.

When a Markush group or other grouping is used herein, all individualmembers of the group and all combinations and possible subcombinationsof the group are intended to be individually included in the disclosure.Every combination of components or materials described or exemplifiedherein can be used to practice the disclosure, unless otherwise stated.One of ordinary skill in the art will appreciate that methods, deviceelements, and materials other than those specifically exemplified can beemployed in the practice of the disclosure without resort to undueexperimentation. All art-known functional equivalents, of any suchmethods, device elements, and materials are intended to be included inthis disclosure. Whenever a range is given in the specification, forexample, a temperature range, a frequency range, a time range, or acomposition range, all intermediate ranges and all sub-ranges, as wellas, all individual values included in the ranges given are intended tobe included in the disclosure. Any one or more individual members of arange or group disclosed herein can be excluded from a claim of thisdisclosure. The disclosure illustratively described herein suitably canbe practiced in the absence of any element or elements, limitation orlimitations which are not specifically disclosed herein.

A number of embodiments of the disclosure have been described. Thespecific embodiments provided herein are examples of useful embodimentsof the disclosure and it will be apparent to one skilled in the art thatthe disclosure can be carried out using a large number of variations ofthe devices, device components, methods steps set forth in the presentdescription. As will be obvious to one of skill in the art, methods anddevices useful for the present methods can include a large number ofoptional composition and processing elements and steps.

In particular, it will be understood that various modifications may bemade without departing from the spirit and scope of the presentdisclosure. Accordingly, other embodiments are within the scope of thefollowing claims.

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The invention claimed is:
 1. A method for interfering with viability ofphenazine-producing bacteria, the method comprising reducing the amountof phenazine in the phenazine-producing bacteria to reduce survivabilityand/or antibiotic resistance of the phenazine-producing bacteria;wherein the reducing comprises degrading phenazine in the bacteria byproviding the phenazine-producing bacteria with one or morephenazine-degrading proteins from Mycobacteria or Streptomyces.
 2. Themethod of claim 1, wherein the reducing further comprises inhibiting thesynthesis of phenazine.
 3. The method of claim 2, wherein the inhibitingsynthesis of phenazine is performed by inactivating one or morephenazine biosynthetic genes.
 4. The method of claim 3, wherein the oneor more phenazine biosynthetic genes are selected from the groupconsisting of PhzA, PhzB, PhzC, PhzD, PhzE, PhzF, PhzF1, PhzF2, PhzG,PhzG1, PhzG2, PhzM, PhzH, and PhzS.
 5. The method of claim 3, whereinthe inhibiting synthesis of phenazine is performed by using smallinterfering RNA techniques to suppress the expression of one or moreproteins in one or more phenazine biosynthetic pathways.
 6. The methodof claim 1, wherein the reducing the amount of phenazine in thephenazine-producing bacteria further comprises chemically modifyingphenazines.
 7. The method of claim 1, wherein the degrading phenazineoccurs exogenously.
 8. The method of claim 1, wherein the one or morephenazine-degrading proteins degrade phenazine-1-carboxylic acid (PCA).9. The method of claim 1, wherein the phenazine-producing bacteria isPseudomonas aeruginosa.
 10. The method of claim 1, wherein thephenazine-producing bacteria is in a biofilm formation.
 11. The methodof claim 1, wherein the phenazine is pyocyanin.
 12. The method of claim1, wherein the phenazine is phenazine-1-carboxylic acid (PCA).